Glycosylated Polypeptide and Drug Composition Containing said Polypeptide

ABSTRACT

[Problem] To provide a glycosylated polypeptide having an affinity to somatostatin receptors, and, compared to somatostatins, having improved in-blood stability. [Solution] The glycosylated polypeptide is characterized by at least one amino acid in a somatostatin or an analogue thereof being replaced with a glycosylated amino acid.

STATEMENT OF PRIORITY

This application is a 35 U.S.C. §371 national phase application ofInternational Application Serial No. PCT/JP2012/072380, filed Sep. 3,2012, which claims the benefit, under 35 U.S.C. §119 (a) of JapanesePatent Application No. 2011-192202, filed Sep. 4, 2011, the entirecontents of each of which are incorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 9778-8TS_ST25v2.txt, 90,691 bytes in size, generated onSep. 9, 2015 and filed via EFS-Web, is provided in lieu of a paper copy.This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

TECHNICAL FIELD

The present invention relates to a glycosylated polypeptide and apharmaceutical composition comprising said polypeptide.

BACKGROUND ART

Somatostatin is a cyclic peptide present in both the central nervoussystem and the surrounding tissue. Somatostatin was first isolated frommammalian hypothalamus, and was identified from anterior pituitary glandas an important inhibitor of growth hormone secretion. This peptide iswidely distributed in e.g. the hypothalamus, the pancreas, and thegastrointestinal tract, and its action is exerted via binding to asomatostatin receptor. In addition, somatostatin is known for itssecretory suppression of growth hormone (GH) and thyroid-stimulatinghormone (TSH) in the pituitary gland, as well as secretion suppressionof various hormones such as gastrin, selectin, cholecystokinin (CCK),and VIP (Vasoactive Intestinal Polypeptide) in the gastrointestinaltract, and glucagon and insulin in the pancreas. It is also known tohave an action to suppress gastrointestinal motility.

Natural somatostatin having the structural formula:Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys (SEQ ID NO. 1)(also known as somatotropin release inhibiting factor (SRIF)) was firstisolated by Guillemin and coworkers. This somatostatin exerts its effectby interacting with a family of receptors. Somatostatin receptor (SSTR)has 1 to 5 subtypes (SSTR1-SSTR5), and among these, SSTR2 is known to bedistributed in each tissues of the GH-secreting human pituitary glandadenoma, the central nervous system, the anterior pituitary gland, theretina, the adrenal medulla, the stomach, the duodenal mucosa, the smallintestines, and the colon, as well as in the glucagon-secreting A-cellof the pancreatic islet. Each of these receptors is also known to beexpressed in various tumors. For example, it has been reported thatSSTR1 and SSTR5 are expressed in a functional pituitary adenoma, SSTR2as well as SSTR1 and SSTR3 are expressed in a gastrointestinal tumor,SSTR3 is expressed in a pheochromocytoma, SSTR1 and SSTR5 are expressedin a prostate cancer, and SSTR5 is expressed in a colorectal cancer(Non-Patent Literature 1). Moreover, SSTR4 is reported in regards to itsfunction as a receptor having an antagonistically modulating action andits possibility of being essential in the treatment of aglaucoma-related disease (Non-Patent Literature 2). As such,somatostatin and analogs thereof are potentially useful therapeuticdrugs for somatostatin-related diseases or various types of tumors.

Meanwhile, because naturally-occurring somatostatin has a shorthalf-life in blood of 2-3 minutes, it shows two undesirable propertiesof having low bioavailability and short duration of action, and thus itsuse or application as a therapeutic is limited. For this reason, varioussomatostatin analogs have been developed in order to find a somatostatinanalog superior in any one of efficacy, biostability, duration ofaction, or selectivity considering the release suppression of growthhormone, insulin, or glucagon.

Octreotide (Patent Literatures 1 and 2) is reported as the firstapproved somatostatin analog that can be clinically utilized, and thisoctreotide is known to have affinity towards somatostatin receptorsSSTR2, SSTR3, and SSTR5.

Octreotide has been developed as a cyclic peptide consisting of eightamino acids which has a sequence of four amino acids (Phe-Trp-Lys-Thr)(SEQ ID NO. 161) that is an important portion for showing the biologicalactivity of somatostatin, Cys that forms a disulfide (S—S) bond at thetwo terminals of the sequence, and further D-Phe and Thr(ol) outside ofthe Cys at the two terminals. This octreotide can render persistence ofaction by improving the half-life in blood by its amino acid sequence,as well as has a higher selectivity towards growth hormone (GH) thansomatostatin which enables it to have a strong action.

Such somatostatin analogs including octreotide can be used for treatingpatients who have a hormone-secreting and hormone-dependent tumor.Currently, symptoms related to metastatic carcinoid tumor which is atumor of the neuroendocrine system (flushing, diarrhea, cardiac valvedisease, and abdominal pain) and symptoms related to vasoactiveintestinal peptide (VIP)-secreting adenoma (watery diarrhea) are treatedwith octreotide.

For example, in a carcinoid and VIP-producing tumor, octreotide inhibitsboth secretion and action of its active factor. Accordingly, in aVIP-producing tumor characterized in profusely-secreting diarrhea, asomatostatin analog can reduce its diarrhea by secretory inhibition ofVIP as well as by directly influencing intestinal secretion.

On the other hand, however, many neuroendocrine tumors are reported tohave resistance to somatostatin analogs such as octreotide (Non-PatentLiterature 3). Moreover, although octreotide is used in the treatment ofacromegaly, it is reported to have no effect on approximately one thirdof acromegaly patients. Further, it is reported that in majority ofcarcinoid tumor patients, octreotide exerts its effect only duringinitial administration, and tachyphylaxis is caused when the duration ofadministration is prolonged. Further, it is reported that octreotidedoes not show any effect on suppression of adrenocorticotropic hormone(ACTH) production in early Cushing's disease patients.

In light of the problems above, development of a somatostatin analogthat binds to multiple receptor subtypes with high affinity like that ofa natural somatostatin is desired for a tumor expressing multiplesomatostatin receptors, and it is suggested that a somatostatin analoghaving such affinity towards somatostatin receptors may possibly alsohave effect on patients who were therapeutically ineffective with orpatients who have resistance to past somatostatin analogs (Non-PatentLiterature 4).

Accordingly, development of a somatostatin analog having a structuresimilar to a naturally-occurring somatostatin, similarly having affinitytowards somatostatin receptors, and having extended half-life in bloodcompared to somatostatin has been desired.

Meanwhile, it has been becoming clear that sugar chains are responsiblefor various roles in vivo, and a method for adding a sugar chain tooctreotide in order to extend the half-life in blood has also beenproposed (such as Patent Literature 3).

However, research is delayed due to the complexity or diversity of itsstructure, and it cannot be said that the type of sugar chain or theposition for adding a sugar chain is always optimized. A glycosylatedpolypeptide that has overcome the problems of past somatostatin analogshas not been reported.

CITATION LIST Patent Literatures

-   [Patent Literature 1] U.S. Pat. No. 4,310,518-   [Patent Literature 2] U.S. Pat. No. 4,235,886-   [Patent Literature 3] Japanese Published Unexamined Patent    Application Publication No. Hei 03 (1991)-014599

Non-Patent Literatures

-   [Non-Patent Literature 1] Current Opinion in Pharmacology, 2004,    Vol. 4, pp. 608-613-   [Non-Patent Literature 2] J. Med. Chem. 2003, Vol. 46, pp. 5587-5596-   [Non-Patent Literature 3] Mol. Endocrinol., 2010, Vol. 24 (1), pp.    240-249-   [Non-Patent Literature 4] Molecular and Cellular Endocrinology, Vol.    286, 2008, pp. 69-74

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The object of the present invention is to provide a glycosylatedpolypeptide having affinity towards somatostatin receptors and improvedstability in blood compared to somatostatin.

Means for Solving the Problems

As a result of repeated research to solve the above problems, thepresent inventors found a glycosylated polypeptide which maintainsaffinity towards somatostatin receptors and has improved stability inblood.

In other words, the present invention relates to a glycosylatedpolypeptide selected from the group consisting of: (A) SRIF14 consistingof the amino acid sequence represented by SEQ ID NO. 1; (B) apolypeptide having one or a few amino acids deleted, substituted, oradded from/to SRIF14 consisting of the amino acid sequence representedby SEQ ID NO. 1; (C) a SRIF14 analog; (D) a polypeptide having 80% ormore homology with SRIF14 consisting of the amino acid sequencerepresented by SEQ ID NO. 1; (E) a polypeptide further comprising Namino acids (wherein N is an integer from 1 or more to 20 or less) atthe N-terminal side of (A)-(D); and (F) a polypeptide further comprisingM amino acids (wherein M is an integer from 1 or more to 6 or less) atthe C-terminal side of (A)-(D); characterized in that at least one aminoacid is substituted with glycosylated amino acid, and the polypeptidehas affinity towards somatostatin receptors.

Here, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that the amino acid substituted with saidglycosylated amino acid is the amino acid corresponding to position 19of SRIF14.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat at least one amino acid is substituted with glycosylated amino acidin said polypeptide (E), and at least one of the amino acids substitutedwith said glycosylated amino acid is present at any of said N aminoacids at the N-terminal side of said polypeptide (E).

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat at least one amino acid is substituted with glycosylated amino acidin said polypeptide (F), and at least one of the amino acids substitutedwith said glycosylated amino acid is present at any of said M aminoacids at the C-terminal side of said polypeptide (F).

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat at least one amino acid is substituted with glycosylated amino acidin said polypeptide (E), and further the sequence of said N amino acidsadded onto the N-terminal side is represented by X—Y—, wherein X means asequence of any L amino acids (wherein L is an integer from 1 or more to6 or less), and Y is a sequence selected from the group consisting of:(1) Lys, (2) Arg-Lys, (3) Glu-Arg-Lys, (4) Arg-Glu-Arg-Lys (SEQ ID NO.162), (5) Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 163), (6)Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 164), (7)Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 165), (8)Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 166), (9)Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 167), (10)Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 168), (11)Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 169), (12)Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 170), (13)Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO. 171),(14) Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys (SEQ ID NO.172), and (15) a sequence having one or a few amino acids deleted,substituted, or added from/to the above sequences (2)-(14).

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that at least one amino acid substitutedwith said glycosylated amino acid is present in any of L amino acidswhich means X in X—Y— representing the sequence of said N amino acidsadded onto the N-terminal side of said polypeptide (E).

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that the N amino acids added onto theN-terminal side are linked to said N-terminal side via a linker.

Moreover, in another embodiment of the glycosylated polypeptide of thepresent invention, a polypeptide selected from the group consisting of:(A) SRIF28 consisting of the amino acid sequence represented by SEQ IDNO. 2; (B) a polypeptide having one or a few amino acids deleted,substituted, or added from/to SRIF28 consisting of the amino acidsequence represented by SEQ ID NO. 2; (C) a SRIF28 analog; (D) apolypeptide having 80% or more homology with SRIF28 consisting of theamino acid sequence represented by SEQ ID NO. 2; (E) a polypeptidefurther comprising J amino acids (wherein J is an integer from 1 or moreto 6 or less) at the N-terminal side of (A)-(D); and (F) a polypeptidefurther comprising K amino acids (wherein K is an integer from 1 or moreto 6 or less) at the C-terminal side of (A)-(D); is characterized inthat at least one amino acid is substituted with glycosylated aminoacid, and the polypeptide has affinity towards somatostatin receptors.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that the amino acid substituted with saidglycosylated amino acid is at least one amino acid selected from thegroup consisting of the amino acid corresponding to position 1, theamino acid corresponding to position 5, the amino acid corresponding toposition 9, the amino acid corresponding to position 12, the amino acidcorresponding to position 13, the amino acid corresponding to position14, and the amino acid corresponding to position 19 of SRIF28.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat at least one amino acid is substituted with glycosylated amino acidin said polypeptide (E), wherein at least one of the amino acidssubstituted with said glycosylated amino acid is present in any of saidJ amino acids at the N-terminal side of said polypeptide (E).

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat at least one amino acid is substituted with glycosylated amino acidin said polypeptide (F), and at least one of the amino acids substitutedwith said glycosylated amino acid is present in any of said K aminoacids of the C-terminal side of said polypeptide (F).

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that said affinity towards somatostatinreceptors has affinity towards at least two or more receptors selectedfrom the group consisting of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat it has affinity towards any one of at least SSTR1 and SSTR4.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat it has affinity towards both SSTR1 and SSTR4.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat it has affinity towards all of SSTR1, SSTR2, SSTR3, SSTR4, andSSTR5.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat it has increased stability in blood compared to SRIF28.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that each of said glycosylated amino acidsis glycosylated Asn or glycosylated Cys.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that in each of said glycosylated aminoacids, the sugar chain and the amino acid are linked without a linker.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that in each of said glycosylated aminoacids, the sugar chain consists of 4 or more sugars.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that in each of said glycosylated aminoacids, the sugar chain is a biantennary complex-type sugar chain, atriantennary complex-type sugar chain, or a tetraantennary complex-typesugar chain.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that in each of said glycosylated aminoacids, the sugar chain is a biantennary complex-type sugar chain.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that said biantennary complex-type sugarchain is a sugar chain selected from the group consisting of a disialosugar chain, a monosialo sugar chain, an asialo sugar chain, a diGlcNAcsugar chain, and a dimannose sugar chain.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that in each of said glycosylated aminoacids, the sugar chain is a sugar chain represented by the followingformula:

[wherein R¹ and R² are identical or different and are:

and Ac is an acetyl group].

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that said sugar chain has at least onesialic acid at the non-reducing terminal, and the carboxy group of saidsialic acid is modified by an alkylamino group, a benzyl group, an aminogroup, or an aminoethylamino group, having 1-30 carbons.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat it has multiple glycosylated amino acids, wherein the sugar chainson each of said glycosylated amino acids are all identical.

Moreover, in one embodiment of the glycosylated polypeptide of thepresent invention, said glycosylated polypeptide is characterized inthat it has Cys corresponding to Cys at position 17 and Cys at position28 of SRIF28, and further these Cys are bound by a disulfide bond witheach other.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that the C-terminal of said glycosylatedpolypeptide is amidated.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that an azido group is introduced at theN-terminal of said glycosylated polypeptide.

Moreover, one embodiment of the glycosylated polypeptide of the presentinvention is characterized in that it is labeled.

Moreover, another aspect of the present invention relates to apharmaceutical composition characterized in that it comprises (I) theglycosylated polypeptide described above and/or a pharmaceuticallyacceptable salt thereof, and (II) a pharmaceutically acceptable carrier.

Moreover, one embodiment of the pharmaceutical composition of thepresent invention is characterized in that the sugar chains in saidglycosylated polypeptide are substantially uniform.

Moreover, one embodiment of the pharmaceutical composition of thepresent invention is characterized in that it is employed for treatmentor prevention of a somatostatin-related disease.

Moreover, one embodiment of the pharmaceutical composition of thepresent invention is characterized in that said somatostatin-relateddisease is at least one disease selected from the group consisting ofacromegaly, gigantism, Alzheimer's disease and other forms of dementia,cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma,insulinoma, glucagonoma, Cushing's disease, hormone secretion defect,diabetes and complications thereof, pains, arthritis, diarrhea, gastriculcer, inflammatory bowel disease, irritable bowel syndrome,gastrointestinal obstruction, ileus, postoperative restenosis, radiationdamage, eye disease, dry eye, glaucoma, interstitial keratitis, iritis,cataract, and conjunctivitis.

Moreover, another aspect of the present invention relates to a methodfor treating or preventing a somatostatin-related disease, characterizedin administering an effective amount of the glycosylated polypeptidedescribed above.

Moreover, one embodiment of the treatment or prophylactic method of thepresent invention is characterized in that said somatostatin-relateddisease is at least one disease selected from the group consisting ofacromegaly, gigantism, Alzheimer's disease and other forms of dementia,cancer, hormone-producing tumor, endocrine tumor, carcinoid, VIPoma,insulinoma, glucagonoma, Cushing's disease, hormone secretion defect,diabetes and complications thereof, pains, arthritis, diarrhea, gastriculcer, inflammatory bowel disease, irritable bowel syndrome,gastrointestinal obstruction, ileus, postoperative restenosis, radiationdamage, eye disease, dry eye, glaucoma, interstitial keratitis, iritis,cataract, and conjunctivitis.

Effects of the Invention

The glycosylated polypeptide of the present invention has affinitytowards somatostatin receptors, and has improved stability in bloodcompared to somatostatin. Moreover, the glycosylated polypeptide of thepresent invention can be employed for treating a somatostatin-relateddisease by virtue of having the above characteristics.

Moreover, since the sugar chain added onto the glycosylated somatostatinof the present invention is easily degraded in vivo, no drug-inducedsuffering is caused to a living body by accumulation thereof.

Moreover, a part or all of the sugar chains added onto the glycosylatedsomatostatin of the present invention is a sugar chain present in theliving body of e.g. mammals and birds including humans or a modifiedversion thereof, and the possibility of showing side effects orantigenicity when administered in vivo is low. There is less concern fore.g. an allergic reactions or antibody production to occur and therebylosing drug effect.

Further, since many of the sugar chains employed herein are relativelyshort, those having uniform structure can be obtained without goingthrough complex manufacturing steps. Accordingly, a pharmaceutical gradehigh quality glycosylated polypeptide having somatostatin activity canbe stably obtained in a large scale.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show each compound name of the glycosylated polypeptides ofan embodiment of the present invention and the amino acid sequenceinformation of the glycosylated polypeptide having said compound name.Moreover, FIG. 1E is a graph showing the IC₅₀ value in regards to cAMPproduction suppressing action (agonist activity) when somatostatinreceptor expression cells were treated with SRIF14, SRIF28, and theglycosylated polypeptides of an embodiment of the present invention.

FIG. 2 is graphs showing the plasma concentration transition of eachpolypeptide when SRIF28 and S1C(disialo)-SRIF28 were administeredintravenously or subcutaneously to rats. In FIG. 2, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 3 is graphs showing the plasma concentration transition whenSRIF28, S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, andS1C(disialo).N5C(disialo)-SRIF28 were administered intravenously andsubcutaneously to rats. In FIG. 3, the graph on the left is a graphshowing the plasma concentration transition of each polypeptide whenadministered intravenously, and the graph on the right is a graphshowing the plasma concentration transition of each polypeptide whenadministered subcutaneously.

FIG. 4 is graphs showing the plasma concentration transition whenSRIF28, S1C(disialo)-SRIF28, S1C(disialo).R13C(disialo)-SRIF28, andS1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 were administeredintravenously and subcutaneously to rats. In FIG. 4, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 5 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28,S1C(disialo).N5C(disialo)-SRIF28, N5C(disialo).A9C(disialo)-SRIF28,S1C(disialo).N5C(disialo)•A9C(disialo)-SRIF28 were administeredintravenously and subcutaneously to rats. In FIG. 5, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 6 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, S1-3C(disialo)-SRIF28 wereadministered intravenously and subcutaneously to rats. In FIG. 6, thegraph on the left is a graph showing the plasma concentration transitionof each polypeptide when administered intravenously, and the graph onthe right is a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 7 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, S1C(disialo(amide))-SRIF28, andS1C(disialo(aminoethylamide))-SRIF28 were administered intravenously andsubcutaneously to rats. In FIG. 7, the graph on the left is a graphshowing the plasma concentration transition of each polypeptide whenadministered intravenously, and the graph on the right is a graphshowing the plasma concentration transition of each polypeptide whenadministered subcutaneously.

FIG. 8 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, S1C(disialo)-D-Trp22-SRIF28, andS1C(disialo(Bn))-SRIF28 were administered intravenously andsubcutaneously to rats. In FIG. 8, the graph on the left is a graphshowing the plasma concentration transition of each polypeptide whenadministered intravenously, and the graph on the right is a graphshowing the plasma concentration transition of each polypeptide whenadministered subcutaneously.

FIG. 9 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, R13C(disialo)-SRIF28, and K14C(disialo)-SRIF28 wereadministered intravenously and subcutaneously to rats. In FIG. 9, thegraph on the left is a graph showing the plasma concentration transitionof each polypeptide when administered intravenously, and the graph onthe right is a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 10 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, E12C(disialo)-SRIF28, N19C(disialo)-SRIF28,29C(disialo)-SRIF28, S1C(monosialo)-SRIF28, and S1C(asialo)-SRIF28 wereadministered intravenously and subcutaneously to rats. In FIG. 10, thegraph on the left is a graph showing the plasma concentration transitionof each polypeptide when administered intravenously, and the graph onthe right is a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 11 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, K14C(disialo)-SRIF28, and C(disialo)-SRIF14 wereadministered intravenously and subcutaneously to rats. In FIG. 11, thegraph on the left is a graph showing the plasma concentration transitionof each polypeptide when administered intravenously, and the graph onthe right is a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 12 is graphs showing the plasma concentration transition whenC(disialo)-SRIF14, C(disialo)-C12 linker-SRIF14, and C(disialo)-PEGlinker-SRIF14 were administered intravenously and subcutaneously torats. In FIG. 12, the graph on the left is a graph showing the plasmaconcentration transition of each polypeptide when administeredintravenously, and the graph on the right is a graph showing the plasmaconcentration transition of each polypeptide when administeredsubcutaneously.

FIG. 13 is graphs showing the plasma concentration transition whenSRIF28, S1C(disialo)-SRIF28, S1C(asialo)-SRIF28, S1C(diGlcNAc)-SRIF28,S1C(diMan)-SRIF28, and S1C(GlcNAc)-SRIF28 were administeredintravenously and subcutaneously to rats. In FIG. 13, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 14 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, S1C(trisialo)-SRIF28, S1C(tetrasialo)-SRIF28,S1-2C(disialo)-SRIF28, and S1C(Asn(disialo))-SRIF28 were administeredintravenously and subcutaneously to rats. In FIG. 14, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 15 is graphs showing the plasma concentration transition whenSRIF28, S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28,S1-3C(disialo)-SRIF28, and S1-4C(disialo)-SRIF28 were administeredintravenously and subcutaneously to rats. In FIG. 15, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 16 is graphs showing the plasma concentration transition whenSRIF14, C(disialo)-SRIF14, C(disialo)-K-SRIF14, C(disialo)-R-K-SRIF14,2C(disialo)-R-K-SRIF14, and 3C(disialo)-R-K-SRIF14 were administeredintravenously and subcutaneously to rats. In FIG. 16, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 17 is graphs showing the plasma concentration transition whenS1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28, and S1-3C(asialo)-SRIF28 wereadministered intravenously and subcutaneously to rats. In FIG. 17, thegraph on the left is a graph showing the plasma concentration transitionof each polypeptide when administered intravenously, and the graph onthe right is a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 18 is graphs showing the plasma concentration transition whenS1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, S1-2C(asialo)-SRIF28,S1-2C(disialo(amide))-SRIF28, S1-2C(disialo(Bn))-SRIF28, andC(disialo(aminoethylamide))-S1C(disialo)-SRIF28 were administeredintravenously and subcutaneously to rats. In FIG. 18, the graph on theleft is a graph showing the plasma concentration transition of eachpolypeptide when administered intravenously, and the graph on the rightis a graph showing the plasma concentration transition of eachpolypeptide when administered subcutaneously.

FIG. 19 is a graph showing the result of plasma stability test employingrat plasma for the glycosylated polypeptides of an embodiment of thepresent invention.

DESCRIPTION OF EMBODIMENTS

A “Somatostatin” herein refers to SRIF14 consisting of a sequence of 14amino acids or SRIF28 consisting of a sequence of 28 amino acids.

In the present specification, the N-terminal Ser in the SRIF28 aminoacid sequence will be set to as position 1, and the C-terminal Cys asposition 28. SRIF14 is in perfect match with the amino acid sequence ofpositions 15 to 28 in the SRIF28 amino acid sequence. Note that position15 of the SRIF28 amino acid sequence is Ala, and the N-terminal Ala inthe SRIF14 amino acid sequence (SEQ ID NO. 1) will be set as position 15in correspondence with position 15 of SRIF28. SRIF14 and SRIF28 have adisulfide bond at Cys at position 17 and Cys at position 28.

SRIF14 has the amino acid sequence below (SEQ ID NO. 1). In the aminoacid sequence below, 15 in “Ala₁₅” means Ala at position 15.

(SEQ ID NO. 1) Ala₁₅-Gly₁₆-Cys₁₇-Lys₁₈-Asn₁₉-Phe₂₀-Phe₂₁-Trp₂₂-Lys₂₃-Thr₂₄-Phe₂₅-Thr₂₆-Ser₂₇-Cys₂₈.

SRIF28 has the amino acid sequence below (SEQ ID NO. 2).

(SEQ ID NO. 2) Ser₁-Ala₂-Asn₃-Ser₄-Asn₅-Pro₆-Ala₇-Met₈-Ala₉-Pro₁₀-Arg₁₁-Glu₁₂-Arg₁₃-Lys₁₄-Ala₁₅-Gly₁₆-Cys₁₇-Lys₁₈-Asn₁₉-Phe₂₀-Phe₂₁-Trp₂₂-Lys₂₃-Thr₂₄-Phe₂₅-Thr₂₆-Ser₂₇-Cys₂₈.

An “amino acid” herein is employed in its broadest meaning, and includesnot only natural amino acids but also non-natural amino acids such asamino acid variants and derivatives. Those skilled in the art willrecognize in light of this broad definition that examples of amino acidsherein include, e.g., natural proteinogenic L-amino acids; D-aminoacids; chemically modified amino acids such as amino acid variants andderivatives; natural non-proteinogenic amino acids such as norleucine,β-alanine, and ornithine; and chemically synthesized compounds havingproperties well-known in the art characteristic of amino acids. Examplesof non-natural amino acids include an α-methylamino acid (such asα-methylalanine), a D-amino acid, a histidine-like amino acid (such as2-amino-histidine, β-hydroxy-histidine, homohistidine,α-fluoromethyl-histidine, and α-methyl-histidine), an amino acid havingexcess methylenes on the side chain (“homo” amino acid), and an aminoacid in which the carboxylic functional group amino acid in the sidechain is substituted with a sulfonate group (such as cysteic acid). Someof the somatostatin analogs having affinity towards somatostatinreceptors are known to comprise a non-natural amino acid. In a preferredaspect, the amino acid contained in the compound of the presentinvention consists only of natural amino acids.

As used herein, when one of the amino acids or a few amino acids aresaid to be deleted, substituted, or added, the number of amino acidssubstituted etc. is not particularly limited as long as affinity towardssomatostatin receptors is retained, but is 1-9, preferably 1-5, and morepreferably approximately 1-3 amino acids, or 20% or less and preferably10% or less of the entire length. The amino acid to be substituted oradded may be a natural amino acid, a non-natural amino acid, or an aminoacid analog, preferably a natural amino acid. Examples of somatostatinpeptides having one of the amino acids or a few amino acids deleted,substituted, or added include, e.g., a somatostatin peptide having Trpat position 22 substituted with a D-form Trp (D-Trp), Asn at position 19deleted (J. Med. Chem., 2001, 44, 2238-2246), Phe at position 25substituted with Tyr, and Met at position 8 substituted with Leu(Endocrinology, 1982, 10:1049-1051).

A “SRIF14 or SRIF28 analog” herein includes a polypeptide structurallysimilar to somatostatin and/or a polypeptide having an overlappingstructure with somatostatin, e.g. a polypeptide having one of the aminoacids or a few amino acids of somatostatin conservatively substituted, amodified somatostatin, a somatostatin fragment having affinity towardssomatostatin receptors, and an elongated somatostatin having affinitytowards somatostatin receptors.

“Having one of the amino acids or a few amino acids conservativelysubstituted” herein refers to an amino acid substitution in which thehydrophylicity and/or hydrophobicity index are similar between theoriginal amino acid and the amino acid to be substituted, and whereinapparent reduction or dissipation of affinity towards somatostatinreceptors before and after such substitution is not caused.

A “modified somatostatin” herein is a modified version of somatostatinincluding a naturally-occurring variant of somatostatin or anartificially modified compound of somatostatin. Examples of suchmodifications include, e.g., alkylation, acylation (such asacetylation), amidation, carboxylation, ester formation, disulfide bondformation, glycosylation, lipidation, phosphorylation, hydroxylation,and binding of a labeling component of one or more amino acid residuesof somatostatin.

A “somatostatin fragment having affinity towards somatostatin receptors”herein is a peptide having one or more amino acids deleted from the N-and/or C-terminals of somatostatin which maintains affinity towardssomatostatin receptors.

An “elongated somatostatin having affinity towards somatostatinreceptors” herein is a peptide having one or more amino acids added tothe N- and/or C-terminals of SRIF28 or SRIF14 which maintains affinitytowards somatostatin receptors.

The glycosylated polypeptide of the present invention comprises aglycosylated polypeptide which is a polypeptide consisting of an aminoacid sequence having 80% or more homology with the amino acid sequencerepresented by SEQ ID NO. 1; a polypeptide consisting of an amino acidsequence having 80% or more homology with the amino acid sequencerepresented by SEQ ID NO. 2; or a polypeptide having an amino acidfurther added to the N- or C-terminal of these polypeptides; wherein atleast one amino acid is substituted with a glycosylated amino acid, andthe polypeptide has affinity towards somatostatin receptors.

A polypeptide having homology with SEQ ID NO. 1 or 2 can have preferably80% or more, 85% or more, 90% or more, and 95% or more homology, as longas it has affinity towards somatostatin receptors.

The glycosylated polypeptide of the present invention also comprises apolypeptide having an amino acid further added to the N- and/orC-terminals of SRIF14 or SRIF28 as described above.

In the glycosylated polypeptide of the present invention, the amino acidfurther added to the N-terminal of SRIF14 is not particularly limited aslong as it maintains affinity towards somatostatin receptors. Forexample, 1 or more to 20 or less amino acids can be added.

Here, the amino acid further added to the N-terminal of SRIF14 can berepresented by X—Y—. Y is an amino acid that binds directly to theN-terminal amino acid of SRIF14 polypeptide, and Y consists of any ofamino acid sequences (1)-(15) below:

(1)  Lys, (2)  Arg-Lys, (3)  Glu-Arg-Lys, (4)  (SEQ ID NO. 162)Arg-Glu-Arg-Lys, (5) (SEQ ID NO. 163) Pro-Arg-Glu-Arg-Lys, (6)(SEQ ID NO. 164) Ala-Pro-Arg-Glu-Arg-Lys, (7) (SEQ ID NO. 165)Met-Ala-Pro-Arg-Glu-Arg-Lys, (8) (SEQ ID NO. 166)Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (9) (SEQ ID NO. 167)Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (10) (SEQ ID NO. 168)Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (11) (SEQ ID NO. 169)Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (12) (SEQ ID NO. 170)Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (13) (SEQ ID NO. 171)Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg- Lys, (14)(SEQ ID NO. 172) Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys,  and (15) a sequence having one or a few amino acidsdeleted, substituted, or added from/to the above sequences (2)-(14).

Moreover, X is any amino acid from 1 or more to 6 or less, and shows any1, 2, 3, 4, 5, or 6 amino acids. Preferably, X is a glycosylated aminoacid, and more preferably glycosylated Asn or glycosylated Cys.

In the glycosylated polypeptide of the present invention, the amino acidfurther added to the N-terminal of SRIF28 is not particularly limited aslong as it maintains affinity towards somatostatin receptors. Forexample, any amino acids from 1 or more to 6 or less can be added, andany 1, 2, 3, 4, 5, or 6 amino acids can be added. The amino acid furtheradded to the N-terminal of SRIF28 is preferably a glycosylated aminoacid, and more preferably glycosylated Asn or glycosylated Cys.Moreover, all of the any 1 or more to 6 or less amino acids can beglycosylated amino acids.

In the glycosylated polypeptide of the present invention, the amino acidfurther added to the C-terminal of SRIF14 or SRIF28 is not particularlylimited as long as it maintains affinity towards somatostatin receptors.For example, any amino acids from 1 or more to 6 or less can be added,and any 1, 2, 3, 4, 5, or 6 amino acids can be added. The amino acidfurther added to the C-terminal of SRIF14 or SRIF28 is preferably aglycosylated amino acid, and more preferably glycosylated Asn orglycosylated Cys. Moreover, all of the any 1 or more to 6 or less aminoacids can be glycosylated amino acids.

A “peptide having one or a few amino acids further added to theN-terminal side of somatostatin (position 1 of SRIF28 or position 15 ofSRIF14)” herein refers to, in the case of SRIR28, those having any aminoacid or glycosylated amino acid further added to the N-terminal Ser atposition 1. Moreover, in the case of SRIF14, it refers to those havingany amino acid or glycosylated amino acid further added to theN-terminal Ala at position 15.

Similarly, a “peptide having one or a few amino acids further added tothe C-terminal side (position 28 of SRIF28 or SRIF14)” refers to thosehaving any amino acid or glycosylated amino acid further added to Cys atposition 28 of SRIF28 or SRIF14.

An amino acid can also be further added to the glycosylated polypeptideof the present invention at its N- or C-terminal via a linker. Examplesof such linkers can include, e.g., an alkyl chain or a polyethyleneglycol chain having amino and carboxy groups at the two terminals sothat it can form a peptide bond with the amino acid. Examples of suchlinkers can include, e.g., —NH—(CH₂)_(n)—CO— (wherein n is an integerand is not limited as long as it does not inhibit the linker function ofinterest, but is preferably an integer from 1-15) or—NH—(CH₂CH₂O)_(m)—CH₂CH₂—CO— (wherein m is an integer and is not limitedas long as it does not inhibit the linker function of interest, but ispreferably an integer from 1-7). More specifically, examples can include—NH—(CH₂)₁₁—CO—(C12 linker) or —NH—(CH₂CH₂O)₃—CH₂CH₂—CO— (PEG linker).Moreover, the amino acid added to the glycosylated polypeptide via alinker is not particularly limited, but is preferably a glycosylatedamino acid. Examples of a glycosylated amino acid can includeglycosylated Asn or glycosylated Cys.

Moreover, in one embodiment of the present invention, an azido group canalso be introduced into the glycosylated polypeptide at its N-terminal.Azidation of the N-terminal of the glycosylated polypeptide is preferredbecause it will allow various molecules to be selectively introduced byutilizing azide-alkyne [3+2] cycloaddition reaction or Staudingerreaction. The method for azidating the glycosylated polypeptide is notparticularly limited, but it can be obtained by e.g. condensing theN-terminal of a glycopeptide synthesized on a resin in solid phasesynthesis and an azido-substituted fatty acid with a condensation agent.Azido-substituted fatty acids can include 4-azidobutanoic acid,5-azido-pentanoic acid, and 6-azidohexanoic acid.

Moreover, in one embodiment of the present invention, a labelingcompound can also be added to the glycosylated polypeptide at itsN-terminal. Examples of the labeling compound used herein can include,but is not limited to, e.g. biotin, fluorescent dyes, and metal ionchelators. The labeling compound can be directly bound to the N-terminalof the glycopeptide, or it can also be bound to the N-terminal of theglycopeptide via a linker. For example, by adding biotin as the labelingcompound to the N-terminal of the glycopeptide, strong binding withavidin can be utilized to enable application as research reagents,clinical test agents, or missile therapy.

The addition of a labeling compound to the glycosylated polypeptide ofthe present invention can be performed by a conventional methodwell-known to those skilled in the art. For example, the N-terminal ofthe glycosylated polypeptide on the resin in solid phase synthesis and alabeling compound can be condensed with a condensation agent.

The “glycosylated polypeptide” of the present invention is characterizedin that at least one amino acid is substituted by glycosylated aminoacid.

A “glycosylated polypeptide” herein includes e.g. a polypeptide havingat least one amino acid of somatostatin substituted with glycosylatedamino acid and a polypeptide having at least one amino acid substitutedwith glycosylated amino acid in the above somatostatin analog, each ofwhich is included in the glycosylated polypeptide of the presentinvention even if one of the amino acids or a few amino acids other thanthe glycosylated amino acid is further deleted, substituted, or added. Apeptide having at least one amino acid substituted with glycosylatedamino acid in a peptide in which the C-terminal thereof is amidated(such as SRIF14NH₂ having the amino acid sequence ofAla-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-NH₂ (SEQ ID NO.3) or SRIF28NH₂ having the amino acid sequence ofSer-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-NH₂(SEQ ID NO. 4) is also included in the glycosylated polypeptide of thepresent invention. Further, a salt of these peptides is also included inglycosylated polypeptides.

A salt as used herein may be any of acid addition salts or base additionsalts. Acids generally employed to form acid addition salts areinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, and phosphoric acid, as well as organic acids suchas p-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid,benzoic acid, and acetic acid. Base addition salts include salts derivedfrom an inorganic base such as ammonium hydroxide or alkali or alkalineearth metal hydroxides, carbonates, and bicarbonates. In particular,pharmaceutically acceptable salts are preferred.

A “glycosylated (sugar chain attached) amino acid” herein is an aminoacid having a sugar chain bound thereto, and the sugar chain and theamino acid may be bound via a linker. The binding site of the sugarchain and the amino acid is not particularly restricted, but the aminoacid is preferably bound to the reducing terminal of the sugar chain.

The type of amino acid to which the sugar chain binds is notparticularly limited, and any of natural amino acids and non-naturalamino acids can be employed. With respect to the glycosylated aminoacids having identical or similar structure as those present asglycopeptides (glycoproteins) in vivo, the glycosylated amino acid ispreferably glycosylated Asn, like N-linked sugar chain as well asglycosylated Ser and glycosylated Thr, and glycosylated Asn, likeO-linked sugar chain is particularly preferred.

Moreover, when the sugar chain and the amino acid are bound via alinker, with respect to easy binding with the linker, the amino acid ofthe glycosylated amino acid is preferably an amino acid having two ormore carboxy groups in a molecule such as aspartic acid and glutamicacid, an amino acid having two or more amino groups in a molecule suchas lysine, arginine, histidine, and tryptophan, an amino acid having ahydroxyl group in the molecule such as serine, threonine, and tyrosine,an amino acid having a thiol group in the molecule such as cysteine, andan amino acid having an amide group in the molecule such as asparagineand glutamine. In particular, with respect to reactivity, aspartic acid,glutamic acid, lysine, arginine, serine, threonine, cysteine,asparagine, and glutamine are preferred.

In any glycosylated polypeptide of the present invention, it is thoughtthat if the sugar chain structure, the structure other than the sugarchain, the number of addition sites for the sugar chain, and the numberof sugar chains added are identical, there is no major difference in thehalf-life in blood of the glycosylated polypeptide of the presentinvention whether the glycosylated amino acid is glycosylated Asn(without a linker) or glycosylated Cys (with a linker).

When the sugar chain and the amino acid are bound via a linker, linkersemployed in the art can be widely used, examples of which can include,e.g., —NH—(CO)—(CH₂)_(a)—CH₂— (wherein a is an integer and is notlimited as long as it does not inhibit the linker function of interest,but is preferably an integer from 0-4), C₁₋₁₀ polymethylene, —CH₂—R—(wherein R is a group produced by having one hydrogen atom detached froma group selected from the group consisting of an alkyl, a substitutedalkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substitutedalkynyl, an aryl, a substituted aryl, a carbocyclic group, a substitutedcarbocyclic group, a heterocyclic group, and a substituted heterocyclicgroup), and —(CO)—(CH₂)_(a)—(CO)— (wherein a is an integer and is notlimited as long as it does not inhibit the linker function of interest,but is preferably an integer from 0-4).

In the glycosylated amino acid, when the sugar chain and the amino acidon the somatostatin backbone are bound via a linker, it is preferredthat the linker also comprises an amino acid at the sugar chainterminal. The type of amino acid is not particularly limited, but apreferred example can include Asn.

The manufacturing method of the glycosylated polypeptide of the presentinvention is not to be limited in any way by a description therefor(such as the description a “glycosylated polypeptide having an aminoacid substituted with a glycosylated amino acid”), and a glycosylatedpolypeptide manufactured with any of methods A or B described below isincluded in the “glycosylated polypeptide having an amino acidsubstituted with a glycosylated amino acid”. Moreover, for example, aglycosylated polypeptide in which a sugar chain without any amino acidbound thereto is bound directly or via a linker to an amino acid on apeptide; a glycosylated polypeptide in which a sugar or a sugar chain isfurther added to the sugar chain added in the glycosylated polypeptidein order to elongate the already added sugar chain; a glycosylatedpolypeptide in which one or a few amino acids are bound to the aminoand/or carboxy group of a glycosylated amino acid, and further linked toone or more somatostatin fragments; and a glycosylated polypeptide inwhich a sugar chain having an amino acid bound thereto is bound via alinker to an amino acid on a peptide are also included in theglycosylated polypeptide of the present invention, as long as the finalstructure matches.

In the glycosylated polypeptide of the present invention, the number ofamino acids to be substituted with a glycosylated amino acid may beappropriately adjusted by e.g. bioactivity such as stability in blood orsecretory suppression of growth hormone etc., the number of amino acidspresent in the final glycosylated polypeptide, or the molecular weightof the glycosylated polypeptide before and after glycosylation. Forexample, it is preferred to substitute 1-10, more preferred tosubstitute 1-5, and even more preferred to replace 1-3 amino acids. Withrespect to simpleness, if the desired activity is obtained by onesubstitution, it is preferred to select one substitution. In one aspectof the present invention, it is preferred to substitute 2 or more, e.g.it is preferred to substitute 2-5, and more preferably to substitute 2-3amino acids. In general, in a glycosylated polypeptide having one aminoacid of somatostatin substituted with a glycosylated amino acid, it istend to increase stability in blood and to decrease affinity towardssomatostatin receptors when one or more of amino acids other than theglycosylated amino acid are further substituted by glycosylated aminoacids. However, on the other hand, the stability in blood of theglycosylated polypeptide will increase, and it is therefore possible tocompensate for or increase the decreased somatostatin activity.

Moreover, when multiple sugar chains are present in a glycosylatedpolypeptide, the each sugar chains can be added to consecutive aminoacids, or can be added to amino acids with intervals in the amino acidsequence of the glycosylated polypeptide. Placing the sugar chainsdensely is preferred because there is no rapid increase in plasmaconcentration. Moreover, with respect to bioavailability, the preferredglycosylation position is adding multiple sugar chains with intervalsrather than adding densely to consecutive amino acids.

In the glycosylated polypeptide of the present invention, the site forsubstituting an amino acid with a glycosylated amino acid canparticularly be adjusted with respect to affinity towards multiplesomatostatin receptors.

In one aspect of the present invention, the site for substituting anamino acid with a glycosylated amino acid can include, with respect tohaving affinity towards multiple receptors in SSTR1-SSTR5 among affinityof the glycosylated polypeptide towards somatostatin receptors, e.g.,the amino acid corresponding to position 19 of SRIF14, the amino acidsadded at the 1st, 2nd, 3rd, 6th, 10th, and 14th positions from theN-terminal side among the amino acids further added to the N-terminalside of SRIF14, and the amino acids added at the 1st and 2nd positionsfrom the C-terminal side among the amino acids further added to theC-terminal side of SRIF14. Preferably, it can include the amino acidsadded at the 3rd, 6th, 10th, and 14th positions from the N-terminal sideamong the amino acids further added to the N-terminal side of SRIF14.

Moreover, similarly with respect to having affinity towards multiplereceptors in SSTR1-SSTR5, it can include the amino acids correspondingto positions 1, 5, 9, 12, 13, 14, and 19 of SRIF28, and the amino acidsadded at the 1st and 2nd positions from the C-terminal side among theamino acids further added to the C-terminal of SRIF28. Preferably, itcan include the amino acids corresponding to positions 1, 5, 9, and 12of SRIF28.

Moreover, particularly, examples of substitution of two or more aminoacids of the glycosylated polypeptide of the present invention withglycosylated amino acids can include, with respect to the glycosylatedpolypeptide having affinity towards multiple somatostatin receptors,e.g., substitutions of the amino acids added at the 10th and 14thpositions from the N-terminal side among the amino acids further addedto the N-terminal side of SRIF14; the amino acids added at the 6th and10th positions from the N-terminal side among the amino acids furtheradded to the N-terminal side of SRIF14; the amino acids added at the 2ndand 14th positions from the N-terminal side among the amino acidsfurther added to the N-terminal side of SRIF14; the amino acids added atthe 14th and 15th positions from the N-terminal side among the aminoacids further added to the N-terminal side of SRIF14; the amino acidsadded at the 14th, 15th, and 16th positions from the N-terminal sideamong the amino acids further added to the N-terminal side of SRIF14;and the amino acids added at the 6th, 10th, and 14th positions from theN-terminal side among the amino acids further added to the N-terminalside of SRIF14. Preferably, it can include, e.g., substitutions of theamino acids added at the 10th and 14th positions from the N-terminalside among the amino acids further added to the N-terminal side ofSRIF14; the amino acids added at the 6th and 10th positions from theN-terminal side among the amino acids further added to the N-terminalside of SRIF14; the amino acids added at the 14th and 15th positionsfrom the N-terminal side among the amino acids further added to theN-terminal side of SRIF14; the amino acids added at the 14th, 15th, and16th positions from the N-terminal side among the amino acids furtheradded to the N-terminal side of SRIF14; and the amino acids added at the6th, 10th, and 14th positions from the N-terminal side among the aminoacids further added to the N-terminal side of SRIF14.

Moreover, similarly, examples of substitution of two or more amino acidswith glycosylated amino acids can include, with respect to theglycosylated polypeptide having affinity towards multiple somatostatinreceptors, e.g., substitutions of the amino acids corresponding topositions 1 and 5; the amino acids corresponding to positions 5 and 9;the amino acids corresponding to positions 1 and 13; the amino acidcorresponding to position 1 and the amino acid added at the 1st positionfrom the N-terminal side among the amino acids further added to theN-terminal side of position 1; the amino acid corresponding to position1 and the amino acids added at the 1st and 2nd positions from theN-terminal side among the amino acids further added to the N-terminalside of position 1; and the amino acids corresponding to positions 1, 5,and 9; preferably, substitutions of the amino acids corresponding topositions 1 and 5; substitutions of the amino acids corresponding topositions 5 and 9; substitution of the amino acid corresponding toposition 1 and the amino acid added at the 1st position among the aminoacids further added to the N-terminal side of position 1; the amino acidcorresponding to position 1 and the amino acids added at the 1st and 2ndpositions from the N-terminal side among the amino acids further addedto the N-terminal side of position 1; and substitutions of the aminoacids corresponding to positions 1, 5, and 9 of SRIF28.

In one aspect of the present invention, the site for substituting anamino acid with a glycosylated amino acid can include, with respect tothe stability in blood of the glycosylated polypeptide, e.g., the aminoacid corresponding to position 19 of SRIF14, the amino acids added atthe 1st, 2nd, 3rd, 6th, 10th, 14th positions from the N-terminal sideamong the amino acids further added to the N-terminal side of SRIF14,and the amino acids added at the 1st and 2nd positions from theC-terminal side among the amino acids further added to the C-terminalside of SRIF14. Preferably, it can include the amino acid correspondingto position 19 of SRIF14, the amino acids added at the 1st and 2ndpositions from the N-terminal side among the amino acids further addedto the N-terminal side of SRIF14, and the amino acids added at the 1stand 2nd positions from the C-terminal side among the amino acids furtheradded to the C-terminal side of SRIF14. More preferably, it is the aminoacid corresponding to position 19 of SRIF14, the amino acid added at the1st position from the N-terminal side among the amino acids furtheradded to the N-terminal side of SRIF14, and the amino acid added at the1st position from the C-terminal side among the amino acids furtheradded to the C-terminal side of SRIF14.

Moreover, similarly with respect to the stability in blood of theglycosylated polypeptide, it is e.g. one or more amino acids selectedfrom the group consisting of the amino acids corresponding to positions1, 5, 9, 12, 13, 14, and 19 of SRIF28 and the amino acids added at the1st and 2nd positions from the C-terminal side among the amino acidsfurther added to the C-terminal side of SRIF28, preferably one or moreamino acids selected from the group consisting of the amino acidscorresponding to positions 13, 14, and 19 of SRIF28 and the amino acidsadded at the 1st and 2nd positions from the C-terminal side among theamino acids further added to the C-terminal side of SRIF28, andparticularly preferably one or more amino acids selected from the groupconsisting of the amino acids corresponding to positions 14 and 19 ofSRIF28 and the amino acid added at the 1st position from the C-terminalside among the amino acids further added to the C-terminal side ofSRIF28. In particular, substitution of an amino acid at a distal sitefrom the N-terminal side of SRIF28 is also preferred.

Here, an “amino acid corresponding to a particular amino acid” refers toan amino acid at the same position corresponding to the amino acidsequence of SRIF14 or SRIF28, as long as there is no addition ordeletion etc. of an amino acid in the glycosylated polypeptide.Moreover, if an addition or deletion of an amino acid is present in theamino acid sequence of a glycosylated polypeptide, it refers to theamino acid at the position that takes into account the shift on theamino acid sequence by the addition or deletion of an amino acid. Forexample, in a glycosylated polypeptide having the sequenceSer₁-Ala₂-Asn₃-Ser₄ (SEQ ID NO:173)-at positions 1 to 4, when one aminoacid (Trp) is added between the amino acids at positions 2 and 3(Ser-Ala-Trp-Asn-Ser-) (SEQ ID NO:174), the amino acid corresponding tothe amino acid at position 3 (Asn) refers to the amino acid (Asn) in theglycosylated polypeptide which has been shifted one to the C-terminalside by the insertion of Trp.

In one aspect of the present invention, the amino acid substituted withan glycosylated amino acid is preferably one or more amino acidsselected from an amino acid other than the amino acids corresponding topositions 17, 21, 22, 23, and 28 of SRIF28, and in particular, it ismore preferably one or more amino acids selected from an amino acidother than the amino acids corresponding to positions 17, 22, 23, and 28of SRIF28.

In one aspect of the present invention, when two or more amino acids aresubstituted with glycosylated amino acids, any combination above can beemployed for the site for substituting an amino acid with a glycosylatedamino acid but is not limited thereto. For example, a combination of onesite being selected from the above preferred sites and other sites beingselected from any site of SRIF14 or SRIF28; or a combination of one sitebeing selected from the above preferred sites and other sites beingselected from any of one or a few amino acids further added to theN-terminal (position 1 of SRIF28 or position 15 of SRIF14) or C-terminalof somatostatin are also included in one preferred aspect of the presentinvention.

In one aspect of the present invention, the site where a deletion,substitution, or addition of an amino acid other than glycosylated aminoacids occurs is preferably e.g. one or more amino acids selected from anamino acid other than the amino acids corresponding to positions 17, 22,23, and 28 of SRIF28.

A “sugar chain” herein refers to a compound made from a string of one ormore unit sugars (monosaccharides and/or derivatives thereof). Whenthere is a string of two or more unit sugars, each unit sugar is boundwith each other by a dehydration condensation with a glycoside bond inbetween. Such sugar chains include, but are not limited to e.g. a widerange such as monosaccharides and polysaccharides contained in vivo(glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine(GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid, and conjugatesand derivatives thereof), as well as a sugar chain degraded or derivedfrom conjugated biomolecules such as degraded polysaccharides,glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids. Thesugar chain may be linear or branched.

Moreover, a “sugar chain” herein also includes a sugar chain derivative,and examples of sugar chain derivatives include, but are not limited to,a sugar chain wherein the sugar constituting the sugar chain is e.g. asugar having a carboxy group (such as aldonic acid in which C-position 1is oxidized to become a carboxylic acid (such as D-gluconic acid whichis oxidized D-glucose) and uronic acid in which the terminal C atom hasbecome a carboxylic acid (D-glucuronic acid which is oxidizedD-glucose)), a sugar having an amino group or an amino group derivative(such as acetylated amino group) (such as N-acetyl-D-glucosamine andN-acetyl-D-galactosamine), a sugar having both amino and carboxy groups(such as N-acetylneuraminic acid (sialic acid) and N-acetylmuramicacid), a deoxylated sugar (such as 2-deoxy-D-ribose), a sulfated sugarcomprising a sulfate group, and a phosphorylated sugar comprising aphosphate group.

A preferred sugar chain herein is a sugar chain that increases thestability in blood of somatostatin and will not dissipate affinitytowards somatostatin receptors when added to somatostatin (whensubstituted with an amino acid of somatostatin in the form of aglycosylated amino acid). In an aspect of the present invention, apreferred sugar chain is a sugar chain that can maintain affinitytowards multiple receptors of somatostatin, and further preferably asugar chain that can maintain affinity towards all the receptors ofsomatostatin when added to somatostatin (when substituted with an aminoacid of somatostatin in the form of a glycosylated amino acid).

The sugar chain in the glycosylated polypeptide of the present inventionis not particularly limited, and may be a sugar chain that exists as aglycoconjugate in vivo (such as a glycopeptide (or a glycoprotein), aproteoglycan, and a glycolipid), or it may be a sugar chain that doesnot exist as a glycoconjugate in vivo.

A sugar chain that exists as a glycoconjugate in vivo is preferred withrespect to the fact that the glycosylated polypeptide of the presentinvention is administered in vivo. Examples of such sugar chains includeN- or O-linked sugar chains which are sugar chains bound to a peptide(or a protein) in vivo as a glycopeptide (or a glycoprotein). AnN-linked sugar chain is preferably employed. N-linked sugar chains caninclude, e.g., a high-mannose form, a complex form, or a hybrid form,particularly preferably a complex form.

Examples of preferred complex-type sugar chains used herein include,e.g., a sugar chain represented by the following general formula:

[wherein R¹ and R² are identical or different and are:

and Ac is an acetyl group].

In the glycosylated polypeptide of the present invention, the sugarchain may be bound to the somatostatin peptide with a method other thanO- and N-linking, even if it is a sugar chain that exists as aglycoconjugate in vivo. For example, as described above, those in whichthe sugar chain is bound to Cys or Lys via a linker are also included inthe glycosylated polypeptide of the present invention.

In one aspect of the present invention, it is preferred that the sugarchain in the glycosylated polypeptide of the present invention is asugar chain consisting of 4 or more, for example, 5 or more, 7 or more,in particular 9 or more, or 11 or more sugars.

In one preferred aspect of the present invention, the sugar chain in theglycosylated polypeptide of the present invention is a sugar chainconsisting of 5-11, 9-11, or 11 sugars.

In one preferred aspect of the present invention, the sugar chain in theglycosylated polypeptide of the present invention is a biantennarycomplex-type sugar chain. A complex-type sugar chain is characterized inthat it comprises two or more types of monosaccharides, and has thebasic structure shown below and a lactosamine structure shown byGa1β1-4GlcNAc.

A biantennary complex-type sugar chain refers to those having amonoantennary sugar chain consisting of 0-3 sugars bound to each of thetwo mannoses at the end of the basic structure. Examples of biantennarycomplex-type sugar chains are preferably e.g. a disialo sugar chain asshown below:

a monosialo sugar chain:

an asialo sugar chain:

a diGlcNAc sugar chain:

and a dimannose sugar chain:

and more preferably a disialo sugar chain.

Moreover, the complex-type sugar chain of the present invention includesnot only the above biantennary complex-type sugar chains, but also atriantennary complex-type sugar chain (triple-branched complex-typesugar chain) and a tetraantennary complex-type sugar chain(quadruple-branched complex-type sugar chain). For example, triantennaryand tetraantennary complex-type sugar chains can include a trisialosugar chain represented by the structural formula below:

and a tetrasialo sugar chain represented by the structural formulabelow:

Moreover, triantennary and tetraantennary complex-type sugar chains canalso include a sugar chain having one or more sugars deleted from thenon-reducing terminal of these trisialo or tetrasialo sugar chains.

Further, the complex-type sugar chain of the present invention includesthose with a fucose added. Complex-type sugar chains with a fucose addedcan include fucose-containing complex-type sugar chains represented bythe structural formula below:

Moreover, a sugar chain having one or more sugars deleted acid from thenon-reducing terminal of these fucose-containing complex-type sugarchains can also be included.

Moreover, a “disialo sugar chain,” a “monosialo sugar chain,” an “asialosugar chain,” a “diGlcNAc sugar chain,” a “dimannose sugar chain,” a“biantennary complex-type sugar chain,” a “triantennary complex-typesugar chain,” a “tetraantennary complex-type sugar chain,” and a“fucose-containing complex-type sugar chain” herein include not onlythose shown in the above chemical formulae, but also those with abinding mode different from examples shown in the chemical formulae, andsuch sugar chains are also preferably employed as a sugar chain of thepresent invention. Examples of such sugar chains include, e.g., adisialo or monosialo sugar chain in which sialic acid and galactose arebound with an (α2->3) bond.

Moreover, when the sugar chain is one having a sialic acid at thenon-reducing terminal of the sugar chain, a sugar chain having thecarboxy group of the sialic acid modified can also be employed. Themodification of the carboxy group of the sialic acid is preferably agroup capable of dissipating the negative charge of the carboxy group orconverting it into positive charge, examples of which can include, e.g.,an alkylamino group, a benzyl group, an amino group, and anaminoethylamino group, having 1-30 carbons. The introduction of thesemodifying groups will dissipate the negative charge of the carboxylgroup of the sialic acid (such as benzyl or amino group) or convert itinto positive charge (such as aminoethylamino group), and it is thuspossible to contemplate blood clearance or control of body distributionof glycosylated polypeptide.

Moreover, the high-mannose sugar chain employed herein is a sugar chainhaving 2 or more mannoses further bound to the basic structure of thecomplex-type sugar chain described above. Because high-mannose sugarchains are bulky, stability in blood may become higher by binding ahigh-mannose sugar chain to the peptide. A sugar chain comprising 5-9mannoses such as a mammalian high-mannose sugar chain is preferred, butit may be a sugar chain comprising more mannoses such as a yeasthigh-mannose sugar chain. Examples of high-mannose sugar chainspreferably employed herein can include, e.g., high-mannose-5 (M-5):

and high-mannose-9 (M-9):

Preferred sugar chains herein can include, e.g., the sugar chainsdescribed below which are a sugar chain having an identical structure (asugar chain in which the type of constituent sugar and the binding modethereof are identical) with a sugar chain that exists in a human body asa glycoprotein bound to a protein (such as a sugar chain described in“FEBS LETTERS Vol. 50, No. 3, February 1975”), or a sugar chain havingone or more sugars deleted from the non-reducing terminal of the same.

In one preferred aspect of the present invention, the sugar chainstructure of the glycosylated amino acids in the glycosylatedpolypeptide of the present invention can be substantially identical, orthey may have different sugar chain structures. When the sugar chainstructure in the glycosylated polypeptide is substantially identical,they are preferably e.g. 90% or more identical or 99% or more identical,and it is most preferable that the sugar chain structure is completelyidentical. As used herein, the sugar chain structure in the glycopeptideis identical refers to the fact that in a glycosylated polypeptidehaving two or more sugar chains added, the type of sugar constitutingthe sugar chain, the binding order, and the binding mode are identicalin the glycopeptide when said sugar chains are compared with each other.Moreover, in one preferred aspect of the present invention, it ispreferred that the sugar chains of the glycosylated polypeptide of thepresent invention are uniform. As used herein, the sugar chains in theglycosylated polypeptide are uniform refers to the fact that theglycosylation sites in the peptide, the type of each sugar constitutingthe sugar chain, the binding order, and the binding mode between sugarsare identical between glycopeptides when sugar chains are comparedbetween glycosylated polypeptides, and that at least 90% or more,preferably 95% or more, and more preferably 99% or more of the sugarchain structure is uniform. In particular, a composition etc. comprisinga glycopeptide in which the sugar chains are uniform betweenglycopeptides has a constant quality, and is preferred particularly infields such as pharmaceuticals manufacture or assays. The proportion ofthe uniform sugar chain can be measured for example by a methodemploying e.g. HPLC, capillary electrophoresis, NMR, and massspectrometry.

The preferred glycosylated polypeptides herein are the glycosylatedpolypeptides (SEQ ID NOs. 5-37, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 110, 112, 114, 116, 118, 120, 123-129, 133-135, 138-141, 148, 155,and 160) manufactured in Examples 1-66 described below. In other words,in the following SRIF28 amino acid sequence:Ser₁-Ala₂-Asn₃-Ser₄-Asn₅-Pro₆-Ala₇-Met₈-Ala₉-Pro₁₀-Arg₁₁-Glu₁₂-Arg₁₃-Lys₁₄-Ala₁₅-Gly₁₆-Cys₁₇-Lys₁₈-Asn₁₉-Phe₂₀-Phe₂₁-Trp₂₂-Lys₂₃-Thr₂₄-Phe₂₅-Thr₂₆-Ser₂₇-Cys₂₈(SEQ ID NO. 2),

(a1) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1 (Example 1) (SEQ ID NO: 5);

(a2) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Asn at position 5 (Example 2) (SEQ ID NO: 6);

(a3) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ala at position 9 (Example 3) (SEQ ID NO: 7);

(a4) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Glu at position 12 (Example 4) (SEQ ID NO: 8);

(a5) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Arg at position 13 (Example 5) (SEQ ID NO: 9);

(a6) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Lys at position 14 (Example 6) (SEQ ID NO: 10);

(a7) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ala at position 15 (Example 7) (SEQ ID NO: 11);

(a8) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Gly at position 16 (Example 8) (SEQ ID NO: 12);

(a9) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Lys at position 18 (Example 9) (SEQ ID NO: 13);

(a10) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Asn at position 19 (Example 10) (SEQ ID NO: 14);

(a11) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Phe at position 21 (Example 11) (SEQ ID NO: 15);

(a12) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Thr at position 26 (Example 12) (SEQ ID NO: 16);

(a13) a glycosylated polypeptide having disialo sugar chain attached Cysfurther added at the C-terminal side of Cys at position 28 (Example 13)(SEQ ID NO: 17);

(a14) a glycosylated polypeptide having Thr-disialo sugar chain attachedCys further added at the C-terminal side of Cys at position 28 (Example14) (SEQ ID NO: 18);

(a15) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having D-form Trp substituted forTrp at position 22 (Example 15) (SEQ ID NO: 19);

(a16) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ala at position 9, and having D-form Trp substituted forTrp at position 22 (Example 16) (SEQ ID NO: 20);

(a17) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having one disialo sugar chainattached Cys further added at N-terminal side (Example 20) (SEQ ID NO:21);

(a18) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1 and Asn at position 5 (Example 21)(SEQ ID NO. 22);

(a19) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1 and Arg at position 13 (Example 22)(SEQ ID NO. 23);

(a20) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Asn at position 5 and Ala at position 9 (Example 23)(SEQ ID NO. 24);

(a21) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having two disialo sugar chainattached Cys further added at the N-terminal side (Example 24) (SEQ IDNO. 25);

(a22) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, Asn at position 5, and Ala atposition 9 (Example 25) (SEQ ID NO. 26);

(a23) a glycosylated polypeptide having monosialo sugar chain attachedCys substituted for Ser at position 1 (Example 26) (SEQ ID NO: 27);

(a24) a glycosylated polypeptide having asialo sugar chain attached Cyssubstituted for Ser at position 1 (Example 27) (SEQ ID NO: 28);

(a25) a glycosylated polypeptide having asialo sugar chain attached Cyssubstituted for Ser at position 1, and having one asialo sugar chainattached Cys further added at the N-terminal side (Example 28) (SEQ IDNO. 29);

(a26) a glycosylated polypeptide having asialo sugar chain attached Cyssubstituted for Ser at position 1, and having two asialo sugar-chainattached Cys further added at the N-terminal side (Example 29) (SEQ IDNO. 30);

(a27) a glycosylated polypeptide having disialo sugar chain attached Asnsubstituted for Asn at position 5 (Example 30) (SEQ ID NO: 31);

(a28) a glycosylated polypeptide having disialo sugar chain attached Asnsubstituted for Ser at position 1 (Example 31) (SEQ ID NO: 32);

(a29) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having GlcNAc-added Cyssubstituted for Asn at position 19 (Example 32) (SEQ ID NO. 33);

(a30) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having dimannose glycosylated Cyssubstituted for Asn at position 19 (Example 33) (SEQ ID NO. 34);

(a31) a glycosylated polypeptide having disialo sugar chain attached Cysfurther added at the N-terminal side of Ser at position 1 (Example 34)(SEQ ID NO. 89);

(a32) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Arg at position 11 (Example 35) (SEQ ID NO: 91);

(a33) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Phe at position 20 (Example 36) (SEQ ID NO: 93);

(a34) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Thr at position 24 (Example 37) (SEQ ID NO: 95);

(a35) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Phe at position 25 (Example 38) (SEQ ID NO: 97);

(a36) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 27 (Example 39) (SEQ ID NO: 99);

(a37) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having Tyr substituted for Phe atposition 25 (Example 41) (SEQ ID NO: 103);

(a38) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1 in which the C-terminal is amidated(Example 42) (SEQ ID NO: 105);

(a39) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1 in which said disialo sugar chainattached Cys is biotinylated (Example 44) (SEQ ID NO: 110);

(a40) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1 in which said disialo sugar chainattached Cys is biotinylated via a PEG linker (Example 45) (SEQ ID NO:112);

(a41) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1 in which an azido group is introducedat the N-terminal of said disialo sugar chain attached Cys (Example 46)(SEQ ID NO: 114);

(a42) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having disialo sugar chainattached Cys substituted for Glu at position 12 (Example 47) (SEQ ID NO:116);

(a43) a glycosylated polypeptide having diGlcNAc sugar chain attachedCys substituted for Ser at position 1 (Example 50) (SEQ ID NO: 123);

(a44) a glycosylated polypeptide having dimannose sugar chain attachedCys substituted for Ser at position 1 (Example 51) (SEQ ID NO: 124);

(a45) a glycosylated polypeptide having dimannnose sugar chain attachedCys substituted for Asn at position 19 (Example 52) (SEQ ID NO: 125);

(a46) a glycosylated polypeptide having N-acetylglucosamine added Cyssubstituted for Ser at position 1 (Example 53) (SEQ ID NO: 126);

(a47) a glycosylated polypeptide having N-acetylglucosamine added Cyssubstituted for Asn at position 19 (Example 54) (SEQ ID NO: 127);

(a48) a glycosylated polypeptide having trisialo sugar chain attachedCys substituted for Ser at position 1 (Example 55) (SEQ ID NO: 128);

(a49) a glycosylated polypeptide having tetrasialo sugar chain attachedCys substituted for Ser at position 1 (Example 56) (SEQ ID NO: 129);

(a50) a glycosylated polypeptide having disialo sugar chain attached Cyshaving a disialo sugar chain modified with an aminoethylamino groupsubstituted for Ser at position 1 (Example 57) (SEQ ID NO: 133);

(a51) a glycosylated polypeptide having disialo sugar chain attached Cyshaving a disialo sugar chain modified with an amino group substitutedfor Ser at position 1 (Example 58) (SEQ ID NO: 134);

(a52) a glycosylated polypeptide having disialo sugar chain attached Cyshaving a disialo sugar chain protected with a benzyl group substitutedfor Ser at position 1 (Example 59) (SEQ ID NO: 135);

(a53) a glycosylated polypeptide having disialo sugar chain attached Cyshaving a disialo sugar chain modified with a hexadecylamino groupsubstituted for Ser at position 1 (Example 60) (SEQ ID NO: 138);

(a54) a glycosylated polypeptide having disialo sugar chain attached Cyshaving a disialo sugar chain modified with an amino group substitutedfor Ser at position 1, and having one disialo sugar chain attached Cyshaving a disialo sugar chain modified with an amino group further addedat the N-terminal side (Example 61) (SEQ ID NO: 139);

(a55) a glycosylated polypeptide having disialo sugar chain attached Cyshaving a disialo sugar chain protected with a benzyl group substitutedfor Ser at position 1, and having one disialo sugar chain attached Cyshaving a disialo sugar chain protected with a benzyl group further addedto the N-terminal side (Example 62) (SEQ ID NO: 140);

(a56) a glycosylated polypeptide having disialo sugar chain attached Asnadded Cys substituted for Ser at position 1 (Example 63) (SEQ ID NO:141);

(a57) a glycosylated polypeptide having disialo sugar chain attached Asnsubstituted for Ser at position 1, and having dimannose added Cyssubstituted for Asn at position 19 (Example 64) (SEQ ID NO: 148);

(a58) a glycosylated polypeptide having disialo sugar chain attached Cyssubstituted for Ser at position 1, and having disialo sugar chainattached Cys having a disialo sugar chain modified with anaminoethylamino group further added at the N-terminal side (Example 65)(SEQ ID NO: 155);

(a59) a glycosylated polypeptide having disialo sugar chain attached Asnsubstituted for Ser at position 1, and having three disialo sugar chainattached Cys further added at the N-terminal side (Example 66) (SEQ IDNO: 160); are preferred.

Moreover, in the SRIF14 amino acid sequence:Ala₁₅-Gly₁₆-Cys₁₇-Lys₁₈-Asn₁₉-Phe₂₀-Phe₂₁-Trp₂₂-Lys₂₃-Thr₂₄-Phe₂₅-Thr₂₆-Ser₂₇-Cys₂₈(SEQ ID NO. 1),

(a60) a glycosylated polypeptide having disialo sugar chain attached Cysfurther added at the N-terminal side of Ala at position 15 (Example 17)(SEQ ID NO: 35);

(a61) a glycosylated polypeptide having disialo sugar chain attachedCys-Arg-Lys- further added at the N-terminal side of Ala at position 15(Example 18) (SEQ ID NO: 36);

(a62) a glycosylated polypeptide having disialo sugar chain attached Cysfurther added at the N-terminal side of Ala at position 15 via a C12linker (Example 19) (SEQ ID NO: 37);

(a63) a glycosylated polypeptide having disialo sugar chain attachedCys-Lys- further added at the N-terminal side of Ala at position 15(Example 40) (SEQ ID NO: 101);

(a64) a glycosylated polypeptide having disialo sugar chain attached Cysfurther added at the N-terminal side of Ala at position 15 via a PEGlinker (Example 43) (SEQ ID NO: 107);

(a65) a glycosylated polypeptide having disialo sugar chain attachedCys-disialo sugar chain attached Cys-Arg-Lys-further added at theN-terminal side of Ala at position 15 (Example 48) (SEQ ID NO. 118);

(a66) a glycosylated polypeptide having disialo sugar chain attachedCys-disialo sugar chain attached Cys-disialo sugar chain attachedCys-Arg-Lys- further added at the N-terminal side of Ala at position 15(Example 49) (SEQ ID NO. 120) are preferred.

The glycosylated polypeptide of the present invention can bemanufactured by integrating a glycosylation step into a peptidesynthesis method well-known to those skilled in the art. A methodutilizing an enzyme represented by transglutaminase can also be employedfor glycosylation, but since there are problems in this case such as theneed for a large amount of the sugar chain to be added, the complicationof purification after the final step, and the restriction forglycosylation position and the sugar chain capable of addition, itcannot be said to be a practical method for large scale manufacturingsuch as pharmaceuticals manufacture though it is possible to employ forsynthesis of a small amount such as for assays.

As specific examples of simple manufacturing methods of the glycosylatedpolypeptide of the present invention which are stable manufacturingmethods of a glycosylated polypeptide having uniform sugar chainstructure, a method for manufacturing a glycosylated polypeptide byusing glycosylated Asn as the glycosylated amino acid and applying awell-known peptide synthesis method such as solid and liquid phasesynthesis (method A), and a method for manufacturing a glycosylatedpolypeptide by manufacturing a peptide in which any amino acid ofsomatostatin is substituted with Cys according to a well-known peptidesynthesis method, and then adding a sugar chain to Cys by chemicalsynthesis (method B) will be exemplified below. Those skilled in the artis able to manufacture various glycosylated polypeptides by referring tothese manufacturing methods, and the glycosylated polypeptide obtainedand the manufacturing method thereof are extremely useful especially inthe field of pharmaceuticals manufacture.

Moreover, these methods A and B can be performed in a combination of twoor more. If it is a synthesis of a small amount such as for assays, itis also possible to further combine the above method employing a sugarchain elongation reaction by a transferase. Method A is described inInternational Publication No. 2004/005330 (US 2005222382 (A1)) andmethod B is described in International Publication No. 2005/010053 (US2007060543 (A1)), the disclosures of which are incorporated herein byreference in their entirety. Moreover, the manufacture of sugar chainshaving uniform sugar chain structure employed in methods A and B aredescribed in e.g. International Publication No. 03/008431 (US 2004181054(A1)), International Publication No. 2004/058984 (US 2006228784 (A1)),International Publication No. 2004/058824 (US 2006009421 (A1)),International Publication No. 2004/070046 (US 2006205039 (A1)), andInternational Publication No. 2007/011055, the disclosures of which areincorporated herein by reference in their entirety.

Method for Manufacturing Glycosylated Polypeptide (Method A)

The glycosylated polypeptide can be manufactured by for example a solidphase synthesis employing glycosylated Asn, the outline of which isshown below.

(1) The carboxy group of an amino acid having the amino group nitrogenprotected with a lipophilic protecting group is bound to a resin. Inthis case, since the amino group nitrogen of the amino acid is protectedwith a lipophilic protecting group, self-condensation between aminoacids is prevented, and the resin reacts with the amino acid to causebinding.

(2) The lipophilic protecting group of the reactant obtained is detachedto form a free amino group.

(3) This free amino group and the carboxy group of any amino acid havingthe amino group nitrogen protected with a lipophilic protecting groupare subjected to amidation reaction.

(4) The above lipophilic protecting group is detached to form a freeamino group.

(5) By repeating the above steps (3) and (4) for once or more, a peptidein which any number of any amino acids are linked and having a resinbound at one end and a free amino group at the other end is obtained.

(6) Finally, by cleaving the resin with an acid, a peptide having thedesired amino acid sequence can be obtained.

In (1), if glycosylated Asn having the amino group nitrogen protectedwith a lipophilic protecting group is employed instead of the amino acidhaving the amino group nitrogen protected with a lipophilic protectinggroup, and the carboxy group of said asparagine portion is reacted withthe hydroxyl group of the resin, a peptide having a glycosylated Asn atthe C-terminal can be obtained.

Moreover, after (2), or after repeating (3) and (4) for any number oftimes that is once or more, if glycosylated Asn having the amino groupnitrogen protected with a lipophilic protecting group is employedinstead of the amino acid having the amino group nitrogen protected witha lipophilic protecting group in (3), a sugar chain can be added at anyposition.

Moreover, by employing glycosylated Asn having the amino group nitrogenprotected with a lipophilic protecting group instead of the amino acidhaving the amino group nitrogen protected with a lipophilic protectinggroup for twice or more times in any of steps (1) and (3), a peptidehaving a sugar chain added at any two or more positions can be obtained.

After binding the glycosylated amino acid, if the lipophilic protectinggroup is detached to form a free amino group, and step (6) is performedimmediately thereafter, a peptide having a glycosylated Asn at theN-terminal can be obtained.

The resin may be a resin generally used in solid phase synthesis, andfor example, 2-chlorotrityl chloride resin (from Merck) functionalizedwith chlorine, Amino-PEGA resin (from Merck) functionalized with anamino group, NovaSyn TGT alcohol resin (from Merck), Wang resin (fromMerck), or HMPA-PEGA resin (from Merck) etc. which have hydroxyl groupscan be employed. Moreover, a linker may exist between the Amino-PEGAresin and the amino acid, and examples of such linkers can include,e.g., 4-hydroxymethylphenoxyacetic acid (HMPA) and4-(4-hydroxymethyl-3-methoxyphenoxy)-butylacetic acid (HMPB).H-Cys(Trt)-Trityl NovaPEG resin (from Merck) etc. in which theC-terminal amino acid is bound to the resin in advance can also beemployed.

Moreover, when the C-terminal is to be amidated, it is preferred toemploy e.g. Rink-Amide-PEGA resin (from Merck) functionalized with anamino group. By cleaving this resin and the peptide with an acid, theC-terminal amino acid of the peptide can be amidated.

In the binding between the resin and the amino acid having the aminogroup nitrogen protected with a lipophilic protecting group, forexample, in order to use a resin having a hydroxyl group or a resinfunctionalized with chlorine, the carboxy group of the amino acid isbound to the resin via an ester bond. Moreover, if a resinfunctionalized with an amino group is to be used, the carboxy group ofthe amino acid is bound to the resin via an amide bond.

The 2-chlorotrityl chloride resin is preferred in that it can preventthe racemization of the terminal Cys when elongating the peptide chainin solid phase synthesis.

Any amino acid can be employed as the amino acid, and examples caninclude the natural amino acids serine (Ser), asparagine (Asn), valine(Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine (Tyr),glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), asparticacid (Asp), glutamic acid (Glu), glutamine (Gln), threonine (Thr),cysteine (Cys), methionine (Met), phenylalanine (Phe), tryptophan (Trp),and proline (Pro).

Moreover, the D-form of the above natural amino acid can also be used.

Examples of lipophilic protecting groups can include, e.g., carbonate-or amide-based protecting groups such as a 9-fluorenylmethoxycarbonyl(Fmoc) group, a t-butyloxycarbonyl (Boc) group, a benzyl group, an allylgroup, an allyloxycarbonyl group, and an acetyl group. When introducinga lipophilic protecting group into an amino acid, e.g. when introducingan Fmoc group, introduction can be carried out by adding9-fluorenylmethyl-N-succinimidyl carbonate and sodium hydrogen carbonateand allowing reaction. The reaction may be performed at 0-50° C.,preferably at room temperature for approximately about 1-5 hours.

As the amino acid protected with a lipophilic protecting group, thosecommercially available can also be used. Examples can includeFmoc-Ser-OH, Fmoc-Asn-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Ile-OH,Fmoc-Ala-OH, Fmoc-Tyr-OH, Fmoc-Gly-OH, Fmoc-Lys-OH, Fmoc-Arg-OH,Fmoc-His-OH, Fmoc-Asp-OH, Fmoc-Glu-OH, Fmoc-Gln-OH, Fmoc-Thr-OH,Fmoc-Cys-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, and Fmoc-Pro-OH.

Moreover, an amino acid protected with a lipophilic protecting groupwherein the protecting group is introduced into the side chain caninclude, e.g., Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Cys(Acm)-OH, Fmoc-Cys(StBu)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Cys(Trt)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, andFmoc-Tyr(tBu)-OH.

Moreover, if it is desired to add a linker in the amino acid sequence ofthe glycosylated polypeptide, the linker can be inserted at a preferredposition by using a linker protected with a lipophilic protecting groupinstead of the above amino acid protected with a lipophilic protectinggroup in the process of solid phase synthesis.

When employing a 2-chlorotrityl chloride resin, esterification can beperformed with a base such as diisopropylethylamine (DIPEA),triethylamine, pyridine, and 2,4,6-collidine. Moreover, when employing aresin having a hydroxyl group, e.g. a well-known dehydrationcondensation agent such as l-mesitylenesulfonyl-3-nitro-1,2,4-triazole(MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide(DIC) can be employed as the esterification catalyst. The proportion ofthe amino acid and the dehydration condensation agent used is 1 part byweight of the former to generally 1-10 parts by weight, preferably 2-5parts by weight of the latter.

The esterification reaction is preferably performed by for exampleplacing the resin in a solid phase column, washing this resin with asolvent, and then adding the amino acid solution. Examples of washingsolvents can include, e.g., dimethylformamide (DMF), 2-propanol, anddichloromethane. Examples of solvents for dissolving the amino acid caninclude, e.g., dimethyl sulfoxide (DMSO), DMF, and dichloromethane. Theesterification reaction may be performed at 0-50° C., preferably at roomtemperature for approximately about 10 minutes-30 hours, preferably forapproximately 15 minutes-24 hours.

It is also preferred to acetylate and cap the unreacted hydroxyl groupson the solid phase at this time with e.g. acetic anhydride.

The detachment of the lipophilic protecting group can be performed byfor example treatment with a base. Examples of bases can include, e.g.,piperidine and morpholine. In such a case, it is preferred that this isperformed in the presence of a solvent. Examples of solvents caninclude, e.g., DMSO, DMF, and methanol.

The amidation reaction of the free amino group with the carboxy group ofany amino acid having the amino group nitrogen protected with alipophilic protecting group is preferably performed in the presence ofan activator and a solvent.

Examples of activators can include, e.g., dicyclohexylcarbodiimide(DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(WSC/HCl), diphenylphosphorylazide (DPPA), carbonyldiimidazole (CDI),diethylcyanophosphonate (DEPC),benzotriazol-1-yloxy-trispyrrolidinophosphonium (DIPCI),benzotriazol-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate(PyBOP), 1-hydroxybenzotriazole (HOBt), hydroxysuccinimide (HOSu),dimethylaminopyridine (DMAP), 1-hydroxy-7-azabenzotriazole (HOAt),hydroxyphthalimide (HOPht), pentafluorophenol (Pfp-OH),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium3-oxide hexafluorophosphate (HCTU),O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphonate (HATU),O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU),and 3,4-dihydro-3-hydrodi-4-oxa-1,2,3-benzotriazine (Dhbt).

The amount of the activator used is preferably 1-20 equivalents,preferably 1-10 equivalents, and further preferably 1-5 equivalents toany amino acid having the amino group nitrogen protected with alipophilic protecting group.

Examples of solvents can include, e.g., DMSO, DMF, and dichloromethane.The reaction may be performed at 0-50° C., preferably at roomtemperature for approximately about 10-30 hours, preferably forapproximately 15 minutes-24 hours. The detachment of the lipophilicprotecting group can be performed similarly to the above.

Treatment with an acid is preferred in order to cleave the peptide chainfrom the resin. Examples of acids can include, e.g., trifluoroaceticacid (TFA) and hydrogen fluoride (HF).

In this way, a glycosylated polypeptide having the desired positionsubstituted with glycosylated Asn can be obtained. Moreover, theglycosylated polypeptide purified as such allows formation of adisulfide bond between deprotected Cys as described below.

In one embodiment of the present invention, when sialic acid iscontained in the non-reducing terminal on the sugar chain ofglycosylated Asn employed in solid phase synthesis, the sialic acid isprevented from being cleaved by the acid treatment. It is thereforepreferred that the carboxy group of said sialic acid is protected by aprotecting group. Examples of protecting groups can include, e.g., abenzyl group, an allyl group, and a diphenylmethyl group. The method forintroduction and detachment of the protecting group can be performed bya well-known method. Moreover, the detachment of the protecting group ispreferably performed after the glycosylated polypeptide manufactured bysolid phase synthesis is cleaved from the resin. After cleaving from theresin, if the glycosylated polypeptide is to be cyclized by allowingformation of a disulfide bond in the glycosylated polypeptide, thedetachment of the protecting group may be before or after the step offorming a disulfide bond.

Method for Manufacturing Glycosylated Polypeptide (Method B)

The glycosylated polypeptide can also be manufactured by a method offirst synthesizing a peptide chain, and then adding a sugar chain to thesynthesized peptide chain. Specifically, a peptide comprising Cys at theposition to be glycosylated is manufactured by e.g. a solid phasesynthesis method, a liquid phase synthesis method, a method of synthesisby a cell, and a method of separating and extracting those thatnaturally exist. Here, Cys that will not be glycosylated such as Cys ata position planned to form a disulfide bond are protected with forexample an acetamidomethyl (Acm) group. Moreover, if Cys that willneither be glycosylated nor be used in the formation of a disulfide bondis to be introduced into the glycosylated polypeptide, Cys can beintroduced by protecting the Cys with a protecting group during theglycosylation and disulfide bond formation steps, and then deprotectingthe same. Such protecting groups can include, e.g., tert-butyl (tBu) or4-methoxybenzyl.

Moreover, when a different sugar chain is to be added to Cys in theglycosylated polypeptide, the different sugar chain can be introduced byfirst unprotecting the Cys where the sugar chain is to be introduced,and then protecting the Cys where a different sugar chain is to beintroduced with e.g. StBu. Specifically, when synthesizing a peptide bye.g. solid phase synthesis, Cys where the first sugar chain is to beintroduced is unprotected, and Cys where the second sugar chain is to beintroduced is made into Cys having a protecting group with e.g.Fmoc-Cys(StBu)-OH. Then, the sugar chain is introduced to theunprotected Cys while retaining the protecting group such as StBu. Next,the StBu group etc. is deprotected to introduce a different sugar chainto the Cys that became unprotected. Note that Cys where the first sugarchain is to be introduced and Cys where the second sugar chain is to beintroduced can be one or more Cys.

In the deprotection of the StBu group, the deprotection can be performedby reaction with a reductant such as tris(2-carboxyethyl)phosphinehydrochloride (TCEP), dithiothreitol (DTT), and tributylphosphine. Theabove reaction may be performed generally at 0-80° C., preferably at5-60° C., and further preferably at 10-35° C. The reaction time ispreferably approximately 30 minutes-5 hours in general. After completionof the reaction, it may be purified with a well-known method (such ashigh performance liquid column chromatography (HPLC)) as appropriate.

When introducing different sugar chains, it is preferred that theintroduction starts from sugar chains more stable against the reductioncondition in the deprotection step of Cys or the acidic condition in thepurification step such as HPLC. In particular, when a sialicacid-containing sugar chain is to be introduced, it is preferred that asugar chain that does not have a sialic acid or a sugar chain havingfewer sialic acid residues is introduced first.

Moreover, if it is desired to add a linker in the amino acid sequence ofthe glycosylated polypeptide, for example, the linker can be inserted ata preferred position of the synthesized polypeptide by using a linkerprotected with a lipophilic protecting group instead of an amino acidprotected with a lipophilic protecting group in the process of solidphase synthesis.

Next, by reacting a haloacetylated complex-type sugar chain derivativewith the peptide comprising an unprotected Cys obtained above, the sugarchain is reacted with the thiol group of the unprotected Cys and boundto the peptide. The above reaction may be performed in a phosphatebuffer, a Tris-hydrochloride buffer, a citrate buffer, or a mixedsolution thereof, generally at 0-80° C., preferably at 10-60° C., andfurther preferably at 15-35° C. The reaction time is generally 10minutes-24 hours, and preferably approximately 30 minutes-5 hours ingeneral. After completion of the reaction, it may be purified with awell-known method (such as HPLC) as appropriate.

A haloacetylated complex-type sugar chain derivative is for example acompound wherein the hydroxyl group bound to the carbon at position 1 ofa complex-type asparagine-linked sugar chain is substituted with—NH—(CH₂)_(a)—(CO)—CH₂X (wherein X is a halogen atom, and a is aninteger and is not limited as long as it does not inhibit the linkerfunction of interest, but is preferably an integer from 0-4).

Specifically, a haloacetylated complex-type sugar chain derivative and aCys-containing peptide are reacted in a phosphate buffer at roomtemperature. After completion of the reaction, a glycosylatedpolypeptide substituted with a glycosylated Cys can be obtained bypurification with HPLC.

Moreover, the reaction can also be performed in a mixed solution of anorganic solvent such as DMSO, DMF, methanol, and acetonitrile with theabove buffer. Here, the organic solvent can be added to the above bufferat a proportion in the range of 0-99% (v/v). For a peptide comprising anunprotected Cys with low solubility to the buffer, addition of such anorganic solvent can improve the solubility to the reaction solution andis thus preferred.

Moreover, the reaction can also be performed in an organic solvent suchas DMSO, DMF, methanol, and acetonitrile or a mixed solution thereof. Inthis case, it is preferably performed in the presence of a base.Examples of bases can include, e.g., DIPEA, triethylamine, pyridine, and2,4,6-collidine. Moreover, the reaction can also be performed in a mixedsolution of guanidine hydrochloride or urea added to the buffersolution. Guanidine hydrochloride or urea can be added to the abovebuffer so that the final concentration will be 1 M-8 M. The solubilityof a peptide with low solubility in the buffer can also be improved byaddition of guanidine hydrochloride or urea and is thus preferred.

Further, in order to prevent the peptide comprising an unprotected Cysfrom forming a dimer via a disulfide bond, tris(2-carboxyethyl)phosphinehydrochloride (TCEP) or dithiothreitol (DTT) can also be added to thebuffer for reaction. TCEP or DTT can be added to the buffer so that thefinal concentration will be 10 μM-10 mM.

Moreover, after binding the sugar chain to the Cys of interest, theprotecting group of Cys protected with Acm etc. is deprotected. When theprotecting group is an Acm group, it can be deprotected by subjecting toreaction with e.g. iodine, mercury acetate (II), silver nitrate (I), orsilver(I) acetate in water, methanol, acetic acid, or a mixed solutionthereof.

The above reaction may be performed generally at 0-80° C., preferably at5-60° C., and further preferably at 10-35° C. The reaction time ispreferably approximately 5 minutes-24 hours in general. After completionof the reaction, it may be treated with DTT or hydrochloric acid etc.,and then purified with a well-known method (such as HPLC) asappropriate.

In this way, a glycosylated polypeptide having the desired positionsubstituted with glycosylated Cys can be obtained. Moreover, theglycosylated polypeptide purified as such allows formation of adisulfide bond between deprotected Cys as described below.

Moreover, in the glycosylated polypeptide prepared by the above methodsA and B, a disulfide bond between Cys can be formed with a methodwell-known to those skilled in the art employing e.g. air and/or oxygen,iodine, DMSO, a mixture of oxidized and reduced glutathione, potassiumferricyanide, Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid)),thallium(III) trifluoroacetate, and alkyltrichlorosilane sulfoxide.

When forming a disulfide bond between Cys-Cys, Cys in the glycosylatedpolypeptide which desirably do not form a disulfide bond is to beprotected with a protecting group. As such protecting groups, aprotecting group which is stable under oxidizing conditions such as Acm,tBu, 4-methoxybenzyl, and 4-methylbenzyl can be employed.

Moreover, in method B, it is also possible to perform the formation ofdisulfide bond before the introduction of the sugar chain. However, if aprotecting group is introduced to Cys that is to be subject to disulfidebonding, the deprotection step will come before the disulfide bondformation step.

(Activity)

The glycosylated polypeptide of the present invention has affinitytowards somatostatin receptors. Having “affinity towards somatostatinreceptors” herein means having affinity towards at least one ofsomatostatin receptors SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5.

The glycosylated polypeptide of the present invention preferably hasaffinity towards two or more somatostatin receptors, more preferably hasaffinity towards three or more receptors, further preferably hasaffinity towards four or more receptors, and most preferably hasaffinity towards all five receptors SSTR1-SSTR5 similarly to a naturalsomatostatin (SRIF28 and SRIF14). In particular, it is preferred that ithas affinity towards any one of at least SSTR1 and SSTR4 and affinitytowards other SSTRs.

As such, the glycosylated polypeptide of the present invention hassomatostatin activity (agonist activity) and antagonist activity towardsa somatostatin receptor by having affinity towards a somatostatinreceptor.

For example, affinity towards each somatostatin receptor can be measuredwith e.g. competitive binding experiment in vitro as shown in Examples67-1 and 67-2.

In the measurement of affinity by competitive binding experiment, theaffinity of the test substance (such as 50% inhibitory concentration:IC₅₀ value or binding inhibition constant: Ki value) can be measured bycompetitively binding a labeled ligand and the test substance to thereceptor and seeking the release of the labeled ligand when the testsubstance is administered.

For example, somatostatin activity (agonist activity) can be evaluatedby a cAMP production suppression test in vitro employing somatostatinreceptor expression cells as shown in Examples 67-3 and 67-4.

The cAMP production suppression test can be evaluated by treatingsomatostatin receptor expression cells with the glycosylated polypeptideor the control compounds SRIF14 or SRIF28, measuring the amount of cAMPaccumulated in the cell after culturing for a certain amount of time,and comparing with the control compound. The amount of cAMP can bemeasured by a well-known method such as the enzyme immunoassay method(EIA).

For example, somatostatin activity (agonist activity) can be evaluatedby a GH production suppression test in vivo as shown in Examples 86 and87.

The GH production suppression test can be carried out for example asfollows. The glycosylated polypeptide is subcutaneously administered tonon-fasted rats. Rats are administered a GH release enhancer undergeneral anesthesia, and then blood is collected in approximately 5minutes. The collected blood is the plasma sample, and GH concentrationis measured by a well-known method such as the enzyme immunoassay method(EIA). Moreover, as a control, plasma samples are obtained from ratswhich were administered with saline that does not contain glycosylatedpolypeptide, and somatostatin activity can be evaluated by comparing theGH concentrations measured.

In the glycosylated polypeptide of the present invention, even if someextent of attenuation in affinity towards each receptor is triggered byglycosylating its structure, the half-life in blood is extended and as aresult, somatostatin activity equivalent to a naturally-occurringsomatostatin that is not glycosylated (hereinafter may be referred to as“non-glycosylated somatostatin”) can be maintained, and it canpreferably have increased somatostatin activity. In light of thisextension of half-life in blood, the ratio between the Ki value of theglycosylated polypeptide of the present invention against each receptorand the Ki value of unglycosylated SRIF14 is preferably in the range of1000:1-0.3:1, more preferably in the range of 100:1-0.3:1, and furtherpreferably in the range of 10:1-0.3:1, as measured in e.g. the methoddescribed in Examples 67-1 and 67-2.

The stability in blood of the glycosylated polypeptide of the presentinvention is preferably equivalent to a naturally-occurring somatostatin(non-glycosylated somatostatin) or more. Stability in blood can bemeasured with a method well-known to those skilled in the art, and canbe decided with e.g. stability in plasma, half-life in blood, and AUC(drug plasma concentration−area under the time curve) as indicators.Moreover, decrease in renal or hepatic clearance also contributes to anincrease in stability in blood.

The glycosylated polypeptide of the present invention has increasedhalf-life in blood compared to an non-glycosylated SRIF28, and itshalf-life is increased by 4-folds or more, preferably 10-fold or more,more preferably 20-folds or more, and further preferably 30-folds ormore compared to SRIF28, as measured in e.g. the experimental methodshown in Example 68.

Moreover, the glycosylated polypeptide of the present invention haspreferably 4-folds or more, more preferably 10-fold or more, and furtherpreferably 20-folds or more stability in blood compared to SRIF28, asmeasured in e.g. the experimental method shown in Example 85.

(Pharmaceutical Composition)

Next, a pharmaceutical composition containing the glycosylatedpolypeptide of the present invention as the active ingredient will bedescribed.

The pharmaceutical composition containing the glycosylated polypeptideof the present invention as the active ingredient is effective for thetreatment or prevention of somatostatin-related diseases. As describedabove, various actions are known for somatostatin, and diseases relatedto these actions also vary. For example, examples ofsomatostatin-related diseases include, e.g., acromegaly, gigantism,Alzheimer's disease and other forms of dementia, cancer,hormone-producing tumor, endocrine tumor (such as carcinoid, VIPoma,insulinoma, and glucagonoma), Cushing's disease, hormone secretiondefect, diabetes and complications thereof, pains, arthritis, diarrhea,gastric ulcer, inflammatory bowel disease, irritable bowel syndrome,gastrointestinal obstruction, ileus, postoperative restenosis, radiationdamage, and eye disease (such as dry eye, glaucoma, interstitialkeratitis, iritis, cataract, and conjunctivitis). The pharmaceuticalcomposition containing the glycosylated polypeptide of the presentinvention as the active ingredient is effective for the treatment orprevention of the above diseases, in particular, acromegaly, dementia,cancer, hormone-producing tumor, endocrine tumor, Cushing's disease,hormone secretion defect, diabetes complication, diarrhea,gastrointestinal obstruction, ileus, radiation damage, eye disease,various tumors or gut-associated disease, and gastrointestinal symptomsaccompanying excess hormone production.

The above pharmaceutical composition is one formulated into the form ofan ordinary pharmaceutical composition with a diluent or an excipientsuch as a generally used filler, expander, binder, moisturizer,disintegrant, surfactant, and lubricant.

Examples of such pharmaceutical compositions include, e.g., tablets,pills, powders, liquids, suspensions, emulsions, granules, capsules,suppositories, inhalants, ophthalmic solutions, and injections.

The amount of the glycosylated polypeptide of the present inventioncontained in the pharmaceutical composition is not particularly limitedand can be selected as appropriate from a broad range, but in general1-70% by weight of the glycosylated polypeptide of the present inventionis preferably contained in the pharmaceutical composition.

The pharmaceutical composition containing the glycosylated polypeptideof the present invention as the active ingredient can either furthercontain other active ingredients, or it can be employed in combinationwith a pharmaceutical composition containing other active ingredients.Moreover, the pharmaceutical composition containing the glycosylatedpolypeptide of the present invention as the active ingredient can eithercomprise the glycosylated polypeptide as a pharmaceutically acceptablesalt, or further contain one or more different glycosylated polypeptidesof the present invention as active ingredients. Moreover, it can also beemployed in combination with the pharmaceutical composition containingone or more different glycosylated polypeptides of the present inventionas the active ingredient. Moreover, Examples of other ingredients thatcan be contained in the pharmaceutical composition can include apharmaceutically acceptable carrier known to those skilled in the art.

Moreover, a treatment using the glycosylated polypeptide of the presentinvention may include e.g. radiation therapy, and it is also useful inscintigraphy for measuring the distribution of cells and tissuesexpressing any of SSTR1-SSTR5 throughout the body. The use ofextracorporeal imaging by radiation scanning or magnetic resonance willenable in vivo semiquantitative detection.

A radiolabeled glycosylated polypeptide is useful for therapeutictreatment of a malignant tumor expressing any of SSTR1-SSTR5, forexample in a human body in a tissue that does not comprise a substantialamount of SSTR1-SSTR5 in a healthy state. Moreover, the labeledglycosylated polypeptide can be administered for scintigraphy, or as acomposition containing an effective amount to suppress a tumor. Examplesof such labels are different isotopes such as iodine (¹²³I, ¹²⁵I, and¹³¹I), indium (¹¹¹In), carbon (¹¹C), fluorine (¹⁸F), technetium (⁹⁹mTc),and yttrium (⁹⁰Y), or a fluorescent label. The labeling can be carriedout by a method well-known to those skilled in the art, and the labelcan be contained in the glycosylated polypeptide, bound directlythereto, or bound to an appropriate compound and then bound to theglycosylated polypeptide. For example, in iodization of tyrosine, thelabeling can be performed by e.g. a method employing chloramine-T etc.

The method for administering the pharmaceutical composition according tothe present invention is not particularly restricted, and it isadministered with a method according to various drug formulations, age,sex, and the disease condition of the patient, and other conditions. Themethod of administration for tablets, pills, liquids, suspensions,emulsions, granules, and capsules include e.g. oral administration.Moreover, in case of injections, it can be intravenously,intramuscularly, intradermally, subcutaneously, or intraperitoneallyadministered alone or mixed with an ordinary fluid replacement such asglucose or amino acid. In case of suppositories, it is intrarectallyadministered. In case of ophthalmic solutions, it is applied to an eyetissue such as the conjunctival sac. In case of inhalants, it is appliedto the bronchial tube or the lung.

The administration dose of the above pharmaceutical composition may beselected as appropriate according to usage, age, sex, and the diseaseextent of the patient, and other conditions, and for example can be anadministration dose that will be 0.1-900 nmol, preferably 1-100 nmol,and more preferably 1-10 nmol of the glycosylated polypeptide of thepresent invention per 1 kg of body weight.

The administration frequency of the above pharmaceutical composition maybe selected as appropriate according to usage, age, sex, and the diseaseextent of the patient, and other conditions, and 3 times/day, twice/day,once/day, and further at a less frequent administration frequencyaccording to stability in blood thereof (such as once/week andonce/month) may be selected. Preferably, the administration frequency ofthe above pharmaceutical composition is once or less per day.

The sugar chain added to the glycosylated polypeptide of the presentinvention is easily degraded by the metabolic system in the body.Moreover, in one aspect of the present invention, said sugar chain has astructure that exists as bound to a glycopeptide (or a glycoprotein) invivo. Accordingly, a pharmaceutical composition comprising theglycosylated polypeptide of the present invention and said peptide asactive ingredients has advantages such as not showing side effects orantigenicity when administered in vivo and less concern for losing drugeffect by allergic reactions or antibody production.

Further, the glycosylated polypeptide of the present invention can bestably and easily supplied in large amounts, and it is extremely usefulwith respect to providing pharmaceuticals having stable and highquality.

Moreover, the present invention also provides a method for treating orpreventing a somatostatin-related disease, characterized inadministering an effective amount of the glycosylated polypeptide of thepresent invention.

The terms used herein are to be employed to describe particularembodiments, and do not intend to limit the invention.

Moreover, the term “comprising” as used herein, unless the contentclearly indicates to be understood otherwise, intends the presence ofthe described items (such as components, steps, elements, and numbers),and does not exclude the presence of other items (such as components,steps, elements, and numbers).

Unless otherwise defined, all terms used herein (including technical andscientific terms) have the same meanings as those broadly recognized bythose skilled in the art of the technology to which the presentinvention belongs. The terms used herein, unless explicitly definedotherwise, are to be construed as having consistent meanings with themeanings herein and related technical fields, and shall not be construedas having idealized or excessively formal meanings.

The embodiments of the present invention may be described referring toschematic diagrams. In case of schematic diagrams, they may beexaggerated in presentation in order to allow clear description.

Terms such as first and second are employed to express various elements,and it should be recognized that these elements are not to be limited bythese terms. These terms are employed solely for the purpose ofdiscriminating one element from another, and it is for example possibleto describe a first element as a second element, and similarly, todescribe a second element as a first element without departing from thescope of the present invention.

The present invention will now be described in further detail referringto Examples. However, the present invention can be embodied by variousaspects, and shall not be construed as being limited to the Examplesdescribed herein.

EXAMPLES

The notation system of the glycosylated polypeptides herein will bedescribed below.

For example, S1C(disialo)-N5C(disialo)-SRIF28 shows that Ser at position1 (S1) and Asn at position 5 of polypeptide SRIF28 are both substitutedby disialo sugar chain attached Cys (C(disialo)).

Moreover, S1C(disialo)-D-Trp22-SRIF28 shows that Ser at position 1 (S1)of polypeptide SRIF28 is substituted by disialo sugar chain attached Cys(C(disialo)), and further Trp at position 22 is substituted with D-Trp.

Moreover, C(disialo)-R-K-SRIF14 shows that glycosylated Cys-Arg-Lys- isadded at the N-terminal side of SRIF14.

Moreover, 29C(disialo)-SRIF28 or 30C(disialo)-SRIF28 shows peptideshaving one disialo sugar chain attached Cys further added to Cys atposition 28 which is the C-terminal of SRIF28 (29C(disialo)-SRIF28), orhaving —W-disialo sugar chain attached Cys (W is any amino acid thatbinds to Cys at position 28) further added to Cys at position 28 whichis the C-terminal of SRIF28 (30C(disialo)-SRIF28).

Moreover, S1-2C(disialo)-SRIF28 shows that Ser at position 1 present atthe N-terminal of polypeptide SRIF28 is substituted with two consecutivedisialo sugar chain attached Cys.

In the meantime, “disialo” means a disialo sugar chain, “monosialo”means a monosialo sugar chain, “asialo” means an asialo sugar chain,“diGlcNAc” means a diGlcNAc sugar chain, “GlcNAc” means anN-acetylglucosamine, “diMan” means a dimannose sugar chain, “trisialo”means a trisialo sugar chain, and “tetrasialo” means a tetrasialo sugarchain. Moreover, (disialo(aminoethylamide)) means that the carboxy groupof the sialic acid of the disialo sugar chain is protected by anaminoethylamino group. “Bn,” “amide,” or “hexadecylamide” instead of“aminoethylamide” each mean that the carboxy group of the sialic acid onthe sugar chain is modified by a benzyl group, an amino group,hexadecylamino group.

Example 1 Synthesis of S1C(Disialo)-SRIF28 1-1 Glycosylation Reaction ofThiol

Peptide 1 (SEQ ID NO. 38) represented by the following formula (1) (fromAPC, Inc.) (60.6 mg, 18.3 μmol) and compound a represented by thefollowing formula (a) (bromoacetamidated oligosaccharide: from OtsukaChemical Co., Ltd.) (85.8 mg, 36.6 μmol, 2.0 equivalents to peptide 1)were dissolved in 33 mM phosphate buffer (pH 7.4, 5.5 mL), and reactedat room temperature for 30 minutes.

The reaction solution was purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous acetic acid (AcOH), B: 0.09% AcOH/10% water/90% acetonitrile,gradient A:B=90:10->75:25, 15 minutes, linear gradient elution] toobtain glycopeptide 2 (SEQ ID NO. 39) represented by the followingformula (2) (60.5 mg, 10.9 μmol, yield 59%).

ESI-MS: (m/z) calcd for C₂₂₉H₃₅₈N₅₀O₁₀₂S₄: [M+3H]³⁺ 1858.3, [M+4H]⁴⁺1394.0, [M+5H]⁵⁺ 1115.4, found 1858.1, 1393.8, 1115.2.

1-2 Deprotection of Acm Group

To glycopeptide 2 obtained in the method described in the above 1-1(51.2 mg, 9.19 μmol) was added an aqueous solution (3.7 mL) of silver(I)acetate (18.8 mg, 113 μmol), and reacted at room temperature for 40minutes. DTT (43.6 mg, 282 μmol) dissolved in 200 mM Tris-HCl buffer (pH7.4, 3.7 mL) and 100 mM ascorbic acid aqueous solution (0.92 mL) wereadded, and this was promptly filtered with a filter. The filtrate waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09%AcOH/10% water/90% acetonitrile, gradient A:B=90:10->75:25, 15 minutes,linear gradient elution] to obtain glycopeptide 3 (SEQ ID NO. 40)represented by the following formula (3) (29.2 mg, 5.38 μmol, yield58%).

ESI-MS: (m/z) calcd for C₂₂₃H₃₄₈N₄₈O₁₀₀S₄: [M+3H]³⁺ 1810.9, [M+4H]⁴⁺1358.4, [M+5H]⁵⁺ 1086.9, [M+ 6H]⁶⁺ 906.0, found 1810.7, 1358.3, 1086.6,905.7.

1-3 Formation of Disulfide Bond

Glycopeptide 3 obtained in the method described in the above 1-2 (29.2mg, 5.38 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 10.8 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=77:23->64:36, 17 minutes, linear gradient elution] to obtain afraction containing compound (S1C(disialo)-SRIF28) represented by thefollowing formula (4) (SEQ ID NO. 5).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=90:10->75:25, 15 minutes, linear gradient elution] to obtainS1C(disialo)-SRIF28 (17.2 mg, 3.17 μmol, yield 59%).

ESI-MS: (m/z) calcd for C₂₂₃H₃₄₆N₄₈O₁₀₀S₄: [M+3H]³⁺ 1810.2, [M+4H]⁴⁺1357.9, [M+5H]⁵⁺ 1086.5, found 1810.0, 1357.5, 1086.4.

Example 2 Synthesis of N5C(Disialo)-SRIF28

A compound represented by the following formula (6)(N5C(disialo)-SRIF28) (SEQ ID NO. 6) was synthesized similarly toExample 1, except that a compound represented by the following formula(5) (peptide 5) (SEQ ID NO. 41) was employed instead of peptide 1.

Example 3 Synthesis of A9C(Disialo)-SRIF28

A compound represented by the following formula (8)(A9C(disialo)-SRIF28) (SEQ ID NO. 7) was synthesized similarly toExample 1, except that a compound represented by the following formula(7) (peptide 7) (SEQ ID NO. 42) was employed instead of peptide 1.

Example 4 Synthesis of E12C(Disialo)-SRIF28

A compound represented by the following formula (10)(E12C(disialo)-SRIF28) (SEQ ID NO. 8) was synthesized similarly toExample 1, except that a compound represented by the following formula(9) (peptide 9) (SEQ ID NO. 43) was employed instead of peptide 1.

Example 5 Synthesis of R13C(Disialo)-SRIF28

A compound represented by the following formula (12)(R13C(disialo)-SRIF28) (SEQ ID NO. 9) was synthesized similarly toExample 1, except that a compound represented by the following formula(11) (peptide 11) (SEQ ID NO. 44) was employed instead of peptide 1.

Example 6 Synthesis of K14C(Disialo)-SRIF28

A compound represented by the following formula (14)(K14C(disialo)-SRIF28) (SEQ ID NO. 10) was synthesized similarly toExample 1, except that a compound represented by the following formula(13) (peptide 13) (SEQ ID NO. 45) was employed instead of peptide 1.

Example 7 Synthesis of A15C(Disialo)-SRIF28

A compound represented by the following formula (16)(A15C(disialo)-SRIF28) (SEQ ID NO. 11) was synthesized similarly toExample 1, except that a compound represented by the following formula(15) (peptide 15) (SEQ ID NO. 46) was employed instead of peptide 1.

Example 8 Synthesis of G16C(Disialo)-SRIF28

A compound represented by the following formula (18)(G16C(disialo)-SRIF28) (SEQ ID NO. 12) was synthesized similarly toExample 1, except that a compound represented by the following formula(17) (peptide 17) (SEQ ID NO. 47) was employed instead of peptide 1.

Example 9 Synthesis of K18C(disialo)-SRIF28

A compound represented by the following formula (20)(K18C(disialo)-SRIF28) (SEQ ID NO. 13) was synthesized similarly toExample 1, except that a compound represented by the following formula(19) (peptide 19) (SEQ ID NO. 48) was employed instead of peptide 1.

Example 10 Synthesis of N19C(Disialo)-SRIF28

A compound represented by the following formula (22)(N19C(disialo)-SRIF28) (SEQ ID NO. 14) was synthesized similarly toExample 1, except that a compound represented by the following formula(21) (peptide 21) (SEQ ID NO. 49) was employed instead of peptide 1.

Example 11 Synthesis of F21C(Disialo)-SRIF28

A compound represented by the following formula (24)(F21C(disialo)-SRIF28) (SEQ ID NO. 15) was synthesized similarly toExample 1, except that a compound represented by the following formula(23) (peptide 23) (SEQ ID NO. 50) was employed instead of peptide 1.

Example 12 Synthesis of T26C(Disialo)-SRIF28

A compound represented by the following formula (26)(T26C(disialo)-SRIF28) (SEQ ID NO. 16) was synthesized similarly toExample 1, except that a compound represented by the following formula(25) (peptide 25) (SEQ ID NO. 51) was employed instead of peptide 1.

Example 13 Synthesis of 29C(Disialo)-SRIF28

A compound represented by the following formula (28)(29C(disialo)-SRIF28) (SEQ ID NO. 17) was synthesized similarly toExample 1, except that a compound represented by the following formula(27) (peptide 27) (SEQ ID NO. 52) was employed instead of peptide 1.

Example 14 Synthesis of 30C(Disialo)-SRIF28

A compound represented by the following formula (30)(30C(disialo)-SRIF28) (SEQ ID NO. 18) was synthesized similarly toExample 1, except that a compound represented by the following formula(29) (peptide 29) (SEQ ID NO. 53) was employed instead of peptide 1.

Example 15 Synthesis of S1C(Disialo)-D-Trp22-SRIF28

A compound represented by the following formula (32)(S1C(disialo)-D-Trp22-SRIF28) (SEQ ID NO. 19) was synthesized similarlyto Example 1, except that a compound represented by the followingformula (31) (peptide 31) (SEQ ID NO. 54) was employed instead ofpeptide 1.

Example 16 Synthesis of A9C(Disialo)-D-Trp22-SRIF28

A compound represented by the following formula (34)(A9C(disialo)-D-Trp22-SRIF28) (SEQ ID NO. 20) was synthesized similarlyto Example 1, except that a compound represented by the followingformula (33) (peptide 33) (SEQ ID NO. 55) was employed instead ofpeptide 1.

Example 17 Synthesis of C(Disialo)-SRIF14

A compound represented by the following formula (36) (C(disialo)-SRIF14)(SEQ ID NO. 35) was synthesized similarly to Example 1, except that acompound represented by the following formula (35) (peptide 35) (SEQ IDNO. 56) was employed instead of peptide 1.

Example 18 Synthesis of C(Disialo)-R-K-SRIF14

A compound represented by the following formula (38)(C(disialo)-R-K-SRIF14) (SEQ ID NO. 36) was synthesized similarly toExample 1, except that a compound represented by the following formula(37) (peptide 37) (SEQ ID NO. 57) was employed instead of peptide 1.

Example 19 Synthesis of C(Disialo)-C12 Linker-SRIF14 19-1 Synthesis ofPeptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF, in a peptide solid phase synthesis method with Fmoc strategyemploying a Prelude™ peptide synthesizer, a protected peptide 39 (SEQ IDNO. 58) represented by the following formula (39) was synthesized in astate bound to the resin. The condensation reaction was performed in DMFusing HCTU as the condensation agent.

The Fmoc protecting group was removed by treating with 20% piperidine inDMF. After washing with DMF and dichloromethane, employing HCTU as thecondensation agent, Fmoc-12-aminododecanoic acid and Fmoc-Cys(Trt)-OHwere condensed in sequence. After condensation, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol(=90:2.5:5:2.5) was added, and this was shaken for 3 hours at roomtemperature. This leads to the detachment of the protecting group of theamino acid side chain (other than the Acm group), as well as cleavingbetween the peptide and the resin. The resin was filtered off, colddiethyl ether was added to the filtrate, and crude peptide was obtainedas precipitate. A part of the crude peptide was purified with HPLC[column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate:7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90%acetonitrile, gradient A:B=60:40->36.2:63.8, 20 minutes, linear gradientelution] to obtain a compound represented by the following formula (40)(peptide 40) (SEQ ID NO. 59) (11.2 mg).

ESI-MS: (m/z) calcd for C₉₇H₁₄₄N₂₂O₂₃S₃: [M+2H]²⁺ 1042.3, [M+3H]³⁺695.2, found 1042.0, 695.0.

19-2 Glycosylation Reaction of Thiol

Peptide 40 obtained in the method described in the above 19-1 (6.8 mg,3.3 μmol) and compound a represented by the above formula (a) (19.1 mg,8.15 μmol) were dissolved in 0.2 M phosphate buffer (pH 7.4, 0.96 mL)containing 7 M guanidine hydrochloride and 330 μM TCEP, and reacted atroom temperature for 2 hours. After confirming the disappearance of rawmaterials with HPLC, the reaction solution was purified with HPLC[column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate:7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90%acetonitrile, gradient A:B=80:20->50:50, 25 minutes, linear gradientelution] to obtain a compound represented by the following formula (41)(glycopeptide 41) (SEQ ID NO. 60) (7.1 mg, 1.6 μmol, yield 50%).

ESI-MS: (m/z) calcd for C₁₈₃H₂₈₃N₂₉O₈₅S₃: [M+3H]³⁺ 1449.5, [M+4H]⁴⁺1087.4, [M+5H]⁵⁺ 870.1, found 1449.3, 1087.2, 870.0.

19-3 Deprotection of Acm Group

To glycopeptide 41 obtained in the method described in the above 19-2(10.3 mg, 2.37 μmol) was added an aqueous solution (0.95 mL) ofsilver(I) acetate (9.7 mg, 58 μmol), and reacted at room temperature for30 minutes. DTT (22.3 mg, 145 μmol) dissolved in 100 mM phosphate buffer(pH 7.4, 0.95 mL) and 100 mM ascorbic acid aqueous solution (0.95 mL)were added, and this was promptly filtered with a filter. The filtratewas purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09%AcOH/10% water/90% acetonitrile, gradient A:B=80:20->50:50, 25 minutes,linear gradient elution] to obtain glycopeptide 42 represented by thefollowing formula 42 (SEQ ID NO. 61) (5.8 mg, 1.4 μmol, yield 59%).

ESI-MS: (m/z) calcd for C₁₇₇H₂₇₃N₂₇O₈₃S₃: [M+3H]³⁺ 1402.1, [M+4H]⁴⁺1051.8, [M+5H]⁵⁺ 841.7, found 1401.9, 1051.7, 841.5.

19-4 Formation of Disulfide Bond

Glycopeptide 42 obtained in the method described in the above 19-3 (5.8mg, 1.4 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 3.5 mL), and reacted at room temperature for 30 hours. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=70:30->50:50, 25 minutes, linear gradient elution] to obtain afraction containing compound (C(disialo)-C12 linker-SRIF14) representedby the following formula 43 (SEQ ID NO. 37).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=80:20->50:50, 25 minutes, linear gradient elution] to obtainC(disialo)-C12 linker-SRIF14 (3.6 mg, 0.86 μmol, yield 61%).

ESI-MS: (m/z) calcd for C₁₇₇H₂₇₁N₂₇O₈₃S₃: [M+3H]³⁺ 1401.5, [M+4H]⁴⁺1051.3, [M+5H]⁵⁺ 841.3, found 1401.2, 1051.2, 841.1.

Example 20 Synthesis of S1-2C(Disialo)-SRIF28 20-1 Synthesis of Peptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF and dichloromethane, in a peptide solid phase synthesis methodwith Fmoc strategy employing a Prelude™ peptide synthesizer, a protectedpeptide was synthesized on a resin. The condensation reaction wasperformed in DMF using HCTU as the condensation agent.

The Fmoc protecting group was removed by treating with 20% piperidine inDMF. After washing with DMF and dichloromethane,TFA:water:triisopropylsilane: ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken for 3 hours at room temperature. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. A part of the crude peptide waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=72:28->68.5:31.5, 20minutes, linear gradient elution] to obtain peptide 44 (SEQ ID NO. 62)represented by the following formula (44) (30.7 mg).

ESI-MS: (m/z) calcd for C₁₄₆H₂₂₄N₄₄O₄₁S₅: [M+3H]³⁺ 1138.3, [M+4H]⁴⁺854.0, [M+5H]⁵⁺ 683.4, found 1138.2, 853.9, 683.1.

20-2 Glycosylation Reaction of Thiol

Peptide 44 obtained in the method described in the above 20-1 (45.8 mg,13.4 μmol) and compound a represented by the above formula (a) (125.8mg, 53.7 μmol, 4.0 equivalents to peptide 44) were dissolved in 33 mMphosphate buffer (pH 7.4, 4.0 mL), and reacted at room temperature for30 minutes. The reaction solution was purified with HPLC [column:SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90%acetonitrile, gradient A:B=83:17->72:28, 15 minutes, linear gradientelution] to obtain glycopeptide 45 represented by the following formula45 (SEQ ID NO. 63) (44.5 mg, 5.61 μmol, yield 42%).

ESI-MS: (m/z) calcd for C₃₁₈H₅₀₂N₅₈O₁₆₅S₅: [M+5H]⁵⁺ 1588.6, [M+6H]⁶⁺1324.0, [M+7H]⁷⁺ 1135.0, [M+8H]⁸⁺ 993.3, [M+9H]⁹⁺ 883.0, found 1588.6,1324.0, 1135.0, 993.2, 883.0.

20-3 Deprotection of Acm Group

To glycopeptide 45 obtained in the method described in the above 20-2(44.5 mg, 5.61 μmol) was added an aqueous solution (2.2 mL) of silver(I)acetate (14.8 mg, 88.7 μmol), and reacted at room temperature for 30minutes. DTT (33.2 mg, 215 μmol) dissolved in 200 mM phosphate buffer(pH 7.4, 2.2 mL) and 100 mM ascorbic acid aqueous solution (561 μL) wereadded, and this was promptly filtered with a filter. The filtrate waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09%AcOH/10% water/90% acetonitrile, gradient A:B=83:17->72:28, 15 minutes,linear gradient elution] to obtain glycopeptide 46 (SEQ ID NO. 64)represented by the following formula (46) (34.4 mg, 4.41 μmol, yield79%).

ESI-MS: (m/z) calcd for C₃₁₂H₄₉₂N₅₆O₁₆₃S₅: [M+4H]⁴⁺ 1950.0, [M+5H]⁵⁺1560.2, [M+6H]⁶⁺ 1300.3, found 1949.8, 1560.1, 1300.2.

20-4 Formation of Disulfide Bond

Glycopeptide 46 obtained in the method described in the above 20-3 (34.4mg, 4.41 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 8.8 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=77:23->65:35, 16 minutes, linear gradient elution] to obtain afraction containing compound (S1-2C(disialo)-SRIF28) represented by thefollowing formula (47) (SEQ ID NO. 21).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=83:17->72:28, 15 minutes, linear gradient elution] to obtainS1-2C(disialo)-SRIF28 (20.1 mg, 2.58 μmol, yield 58%).

ESI-MS: (m/z) calcd for C₃₁₂H₄₉₀N₅₆O₁₆₃S₅: [M+4H]⁴⁺ 1949.5, [M+5H]⁵⁺1559.8, [M+6H]⁶⁺ 1300.0, found 1949.4, 1559.7, 1299.9.

Example 21 Synthesis of S1C(Disialo).N5C(Disialo)-SRIF28

A compound represented by the following formula (49)(S1C(disialo).N5C(disialo)-SRIF28) (SEQ ID NO. 22) was synthesizedsimilarly to Example 20, except that a compound represented by thefollowing formula (48) (peptide 48) (SEQ ID NO. 65) was employed insteadof peptide 44.

Example 22 Synthesis of S1C(Disialo).R13C(Disialo)-SRIF28

A compound represented by the following formula (51)(S1C(disialo).R13C(disialo)-SRIF28) (SEQ ID NO. 23) was synthesizedsimilarly to Example 20, except that a compound represented by thefollowing formula (50) (peptide 50) (SEQ ID NO. 66) was employed insteadof peptide 44.

Example 23 Synthesis of N5C(Disialo).A9C(Disialo)-SRIF28

A compound represented by the following formula (53)(N5C(disialo)-A9C(disialo)-SRIF28) (SEQ ID NO. 24) was synthesizedsimilarly to Example 20, except that a compound represented by thefollowing formula (52) (peptide 52) (SEQ ID NO. 67) was employed insteadof peptide 44.

Example 24 Synthesis of S1-3C(Disialo)-SRIF28 24-1 Synthesis of Peptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF, in a peptide solid phase synthesis method with Fmoc strategyemploying a Prelude™ peptide synthesizer, a protected peptide wassynthesized on the resin. The condensation reaction was performed in DMFusing HCTU as the condensation agent.

The Fmoc protecting group was removed by treating with 20% piperidine inDMF. After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. A part of the crude peptide waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ50×250 mm, flow rate: 43.7 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=73:27->65:35, 14 minutes,linear gradient elution] to obtain peptide 54 (SEQ ID NO. 68)represented by the following formula (54) (41.7 mg).

ESI-MS: (m/z) calcd for C₁₄₉H₂₂₉N₄₅O₄₂S₆: [M+3H]³⁺ 1172.7, [M+4H]⁴⁺879.8, [M+5H]⁵⁺ 704.0, found 1172.5, 879.4, 703.9.

24-2 Glycosylation Reaction of Thiol

Peptide 54 obtained in the method described in the above 24-1 (10.7 mg,3.04 μmol) and compound a represented by the above formula (a) (36.6 mg,15.6 μmol, 5.2 equivalents to peptide 54) were dissolved in 33 mMphosphate buffer (pH 7.4, 0.91 mL) containing 10 μM TCEP, and reacted atroom temperature for 100 minutes. The reaction solution was purifiedwith HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm,flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10%water/90% acetonitrile, gradient A:B=80:20->70:30, 5 minutes, thenA:B=70:30->65:35, 12 minutes, linear gradient elution] to obtainglycopeptide 55 (SEQ ID NO. 69) represented by the following formula(55) (11.7 mg, 1.14 μmol, yield 38%).

ESI-MS: (m/z) calcd for C₄₀₇H₆₄₆N₆₆O₂₂₈S₆: [M+5H]⁵⁺ 2061.8, [M+6H]⁶⁺1718.4, [M+7H]⁷⁺ 1473.0, [M+8H]⁸⁺ 1289.0, [M+9H]⁹⁺ 1145.9, [M+10H]¹⁰⁺1031.4, found 2061.8, 1718.2, 1472.9, 1289.0, 1145.8, 1031.3.

24-3 Deprotection of Acm Group

To glycopeptide 55 obtained in the method described in the above 24-2(11.7 mg, 1.14 μmol) was added an aqueous solution (0.46 mL) ofsilver(I) acetate (4.7 mg, 28 μmol), and reacted at room temperature for2 hours. DTT (11.3 mg, 73 μmol) dissolved in 200 mM Tris-HCl buffer (pH7.4, 0.46 mL) and 100 mM ascorbic acid aqueous solution (0.11 mL) wereadded, and this was promptly filtered with a filter. The filtrate waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09%AcOH/10% water/90% acetonitrile, gradient A:B=80:20->70:30, 5 minutes,then 70:30->55:45, 15 minutes, linear gradient elution] to obtainglycopeptide 56 (SEQ ID NO. 70) represented by the following formula(56) (7.4 mg, 0.73 μmol, yield 64%).

ESI-MS: (m/z) calcd for C₄₀₁H₆₃₆N₆₄O₂₂₆S₆: [M+6H]⁶⁺ 1694.7, [M+7H]⁷⁺1452.7, [M+8H]⁸⁺ 1271.3, [M+9H]⁹⁺ 1130.1, [M+10H]¹⁰⁺ 1017.2, found1694.6, 1452.5, 1271.4, 1130.0, 1017.2.

24-4 Formation of Disulfide Bond

Glycopeptide 56 obtained in the method described in the above 24-3 (7.4mg, 0.73 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 1.8 mL), and reacted at room temperature for 25 hours. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=80:20->70:30, 5 minutes, then 70:30->69.3:30.7, 5 minutes, lineargradient elution] to obtain a fraction containing compound(S1-3C(disialo)-SRIF28) represented by the following formula (57) (SEQID NO. 25).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=80:20->40:60, 20 minutes, linear gradient elution] to obtainS1-3C(disialo)-SRIF28 (4.7 mg, 0.46 μmol, yield 63%).

ESI-MS: (m/z) calcd for C₄₀₁H₆₃₄N₆₄O₂₂₆S₆: [M+3H]³⁺ 3387.7, [M+4H]⁴⁺2541.0, [M+5H]⁵⁺ 2033.0, [M+6H]⁶⁺ 1694.3, [M+7H]⁷⁺ 1452.4, found 3387.6,2540.9, 2032.7, 1694.2, 1452.3.

Example 25 Synthesis of S1C(Disialo).N5C(Disialo).A9C(Disialo)-SRIF28

A compound represented by the following formula (59)(S1C(disialo).N5C(disialo).A9C(disialo)-SRIF28) (SEQ ID NO. 26) wassynthesized similarly to Example 24, except that peptide 58 (SEQ ID NO.71) represented by the following formula (58) was employed instead ofpeptide 54.

Example 26 Synthesis of S1C(Monosialo)-SRIF28

A compound represented by the following formula (60)(S1C(monosialo)-SRIF28) (SEQ ID NO. 27) was synthesized similarly toExample 1, except that compound b represented by the following formula(b) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.)was employed instead of compound a.

In the final product, the ratio between the glycosylated polypeptidehaving the sugar chain of the following formula b1 and the glycosylatedpolypeptide having the sugar chain of the following formula b2 was45:55. Note that it is possible to manufacture a glycosylatedpolypeptide having substantially uniform sugar chain structure by usingmonosialo sugar chain derivatives having identical structure.

Example 27 Synthesis of S1C(Asialo)-SRIF28

A compound represented by the following formula (61)(S1C(asialo)-SRIF28) (SEQ ID NO. 28) was synthesized similarly toExample 1, except that compound c represented by the following formula(c) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.)was employed instead of compound a.

Example 28 Synthesis of S1-2C(Asialo)-SRIF28 28-1 Glycosylation Reactionof Thiol

Peptide 44 (21.2 mg, 6.21 μmol) obtained by the method described in theabove 20-1 and compound c (44.5 mg, 25.3 μmol, 4.1 equivalents topeptide 44) were dissolved in 33 mM phosphate buffer (pH 7.4, 1.9 mL),and reacted at room temperature for 1 hour. The reaction solution waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=77:23->62:38, 15 minutes,linear gradient elution] to obtain glycopeptide 62 (SEQ ID NO. 72)represented by the following formula (62) (24.0 mg, 3.54 μmol, yield57%).

ESI-MS: (m/z) calcd for C₂₇₄H₄₃₄N₅₄O₁₃₃S₅: [M+4H]⁴⁺ 1694.3, [M+5H]⁵⁺1355.6, [M+6H]⁶⁺ 1129.8, found 1694.3, 1355.6, 1130.0.

28-2 Deprotection of Acm Group

To glycopeptide 62 obtained in the method described in the above 28-1(24.0 mg, 3.54 μmol) was added an aqueous solution (1.4 mL) of silver(I)acetate (6.0 mg, 36 μmol), and reacted at room temperature for 3 hours.DTT (14.0 mg, 90.8 μmol) dissolved in 500 mM phosphate buffer (pH 7.4,0.57 mL) and 100 mM ascorbic acid aqueous solution (0.35 mL) were added,and this was promptly filtered with a filter. The filtrate was purifiedwith HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm,flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10%water/90% acetonitrile, gradient A:B=75:25->65:35, 15 minutes, lineargradient elution] to obtain glycopeptide 63 (SEQ ID NO. 73) representedby the following formula (63) (20.1 mg, 3.03 μmol, yield 86%)

ESI-MS: (m/z) calcd for C₂₆₈H₄₂₄N₅₂O₁₃₁S₅: [M+4H]⁴⁺ 1658.7, [M+5H]⁵⁺1327.2, found 1658.8, 1327.0.

28-3 Formation of Disulfide Bond

Glycopeptide 63 obtained in the method described in the above 28-2 (20.1mg, 3.03 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 6.1 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=77:23->65:35, 16 minutes, linear gradient elution] to obtain afraction containing compound (S1-2C(asialo)-SRIF28) represented by thefollowing formula (64) (SEQ ID NO. 29).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=92:8->80:20, 16 minutes, linear gradient elution] to obtainS1-2C(asialo)-SRIF28 (11.0 mg, 1.66 μmol, yield 55%).

ESI-MS: (m/z) calcd for C₂₆₈H₄₂₂N₅₂O₁₃₁S₅: [M+4H]⁴⁺ 1658.2, [M+5H]⁵⁺1326.8, [M+6H]⁶⁺ 1105.8, [M+7H]⁷⁺ 948.0, [M+8H]⁸⁺ 829.6, found 1658.1,1326.7, 1105.6, 947.8, 829.4.

Example 29 Synthesis of S1-3C(Asialo)-SRIF28 29-1 Glycosylation Reactionof Thiol

Peptide 54 (21.3 mg, 6.06 μmol) obtained by the method described in theabove 24-1 and compound c (53.4 mg, 30.3 μmol, 5.0 equivalents topeptide 54) were dissolved in 33 mM phosphate buffer (pH 7.4, 1.8 mL),and reacted at room temperature for 1 hour. The reaction solution waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=75:25->70:30, 20 minutes,linear gradient elution] to obtain glycopeptide 65 (SEQ ID NO. 74)represented by the following formula (65) (39.3 mg, 4.59 μmol, yield76%).

ESI-MS: (m/z) calcd for C₃₄₁H₅₄₄N₆₀O₁₈₀S₆: [M+4H]⁴⁺ 2140.2, [M+5H]⁵⁺1712.3, [M+6H]⁶⁺ 1427.1, found 2140.2, 1712.4, 1427.2.

29-2 Deprotection of Acm Group

To glycopeptide 65 obtained in the method described in the above 29-1(39.3 mg, 4.59 μmol) was added an aqueous solution (1.8 mL) of silver(I)acetate (18.7 mg, 112 μmol), and reacted at room temperature for 90minutes. DTT (43.4 mg, 28.1 μmol) dissolved in 200 mM phosphate buffer(pH 7.4, 1.8 mL) and 100 mM ascorbic acid aqueous solution (0.46 mL)were added, and this was promptly filtered with a filter. The filtratewas purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=75:25->68:32, 18 minutes,linear gradient elution] to obtain glycopeptide 66 (SEQ ID NO. 75)represented by the following formula (66) (27.6 mg, 3.28 μmol, yield71%).

ESI-MS: (m/z) calcd for C₃₃₅H₅₃₄N₅₈O₁₇₈S₆: [M+4H]⁴⁺ 2104.6, [M+5H]⁵⁺1683.9, [M+6H]⁶⁺ 1403.4, found 2104.6, 1684.0, 1403.3.

29-3 Formation of Disulfide Bond

Glycopeptide 66 obtained in the method described in the above 29-2 (27.6mg, 3.28 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 8.2 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=72:28->70.5:29.5, 15 minutes, linear gradient elution] to obtain afraction containing compound (S1-3C(asialo)-SRIF28) represented by thefollowing formula (67) (SEQ ID NO. 30).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=96:4->82:18, 20 minutes, linear gradient elution] to obtainS1-3C(asialo)-SRIF28 (14.5 mg, 1.72 μmol, yield 52%).

ESI-MS: (m/z) calcd for C₃₃₅H₅₃₂N₅₈O₁₇₈S₆: [M+4H]⁴⁺ 2104.1, [M+5H]⁵⁺1683.5, [M+6H]⁶⁺ 1403.1, found 2103.7, 1683.3, 1403.0.

Example 30 Synthesis of N5N(Disialo)-SRIF28 30-1 Solid Phase Synthesisof Glycopeptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Trt)-OH (72.5 mg, 120 μmol) and DIPEA (104.6 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF, in a peptide solid phase synthesis method with Fmoc strategyemploying a Prelude™ peptide synthesizer, a protected peptide 68 (SEQ IDNO. 76) represented by the following formula (68) was synthesized in astate bound to the resin. The condensation reaction was performed in DMFusing HCTU as the condensation agent.

Next, the Fmoc protecting group was removed by treating with 20%piperidine in DMF. After washing with DMF, compound d represented by thefollowing formula (d) (from Otsuka Chemical Co., Ltd.) (411.9 mg, 150.4μmol), DMSO-DMF (1/1, v/v, 871 μL) solution, TBTU (64.2 mg, 200 μmol),and DIPEA (52.3 μL, 300 μmol) were sequentially added to the resin, andthis was shaken at room temperature for 3 hours to allow condensation.

After washing with DMF, this condensation operation was repeated once.After washing the resin with DMF and dichloromethane, this was shakenwith 20% piperidine in DMF for 20 minutes to deprotect the Fmoc group,and the resin was washed with DMF to synthesize a protected compoundrepresented by the following formula (69) (peptide 69) (SEQ ID NO. 77)on the resin.

To this resin, employing HOBt/DIC as the condensation agent,Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ala, and Fmoc-Ser(tBu)-OH werecondensed in sequence. After condensation, the Fmoc protecting group wasremoved by treating with 20% piperidine in DMF. After washing with DMFand dichloromethane, TFA:water:triisopropylsilane:ethanedithiol(=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3hours. Cold diethyl ether was added to the filtrate, and crude peptidewas obtained as precipitate. This crude peptide was purified with HPLC[column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate:7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90%acetonitrile, A:B=70:30] to obtain glycopeptide 70 (SEQ ID NO. 78)represented b the following formula (70) (29.1 mg, 5.26 μmol).

ESI-MS: (m/z) calcd for C₂₃₅H₃₅₇N₄₇O₁₀₀S₃: [M+3H]³⁺ 1846.6, [M+4H]⁴⁺1385.2, [M+5H]⁵⁺ 1108.4, found 1846.5, 1385.1, 1108.3.

30-2 Formation of Disulfide Bond

Glycopeptide 70 obtained in the method described in the above 30-1 (12.2mg, 2.20 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 5.5 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=80:20->66:35, 30 minutes, linear gradient elution] to obtainglycopeptide 71 (SEQ ID NO. 79) represented by the following formula(71) (8.3 mg, 1.5 μmol, yield 68%).

ESI-MS: (m/z) calcd for C₂₃₅H₃₅₅N₄₇O₁₀₀S₃: [M+3H]³⁺ 1846.5, [M+4H]⁴⁺1384.7, [M+5H]⁵⁺ 1108.0, found 1846.5, 1384.7, 1108.1.

30-3 Deprotection of Benzyl Group

Glycopeptide 71 obtained in the method described in the above 30-2 (7.5mg, 1.4 μmol) was dissolved in 50 mM sodium hydroxide aqueous solution(20.6 mL), and reacted at 0° C. for 80 minutes. 200 mM acetic acidaqueous solution (5.1 mL) was added, and the mixed solution was purifiedwith HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm,flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10%water/90% acetonitrile, gradient A:B=73:27->66.3:33.7, 20 minutes,linear gradient elution] to obtain a fraction containing compound(N5N(disialo)-SRIF28) represented by the following formula (72) (SEQ IDNO. 31).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=80:20->60:40, 30 minutes, linear gradient elution] to obtainN5N(disialo)-SRIF28 (1.4 mg, yield 19%).

ESI-MS: (m/z) calcd for C₂₂₁H₃₄₃N₄₇O₁₀₀S₃: [M+3H]³⁺ 1785.9, [M+4H]⁴⁺1339.6, [M+5H]⁵⁺ 1071.9, found 1785.7, 1339.5, 1071.8.

Example 31 Synthesis of S1N(Disialo)-SRIF28 31-1 Solid Phase Synthesisof Glycopeptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Trt)-OH (72.5 mg, 120 μmol) and DIPEA (104.6 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF, in a peptide solid phase synthesis method with Fmoc strategyemploying a Prelude™ peptide synthesizer, a protected peptide 73 (SEQ IDNO. 80) represented by the following formula (73) was synthesized in astate bound to the resin. The condensation reaction was performed in DMFusing HCTU as the condensation agent.

Next, the Fmoc protecting group was removed by treating with 20%piperidine in DMF. After washing with DMF, compound d (420.2 mg, 153.3μmol), DMSO-DMF (1/1, v/v, 871 μL) solution, TBTU (64.2 mg, 200 μmol),and DIPEA (52.3 μL, 300 μmol) were sequentially added to the resin, andthis was shaken at room temperature for 2 hours to allow condensation.After washing with DMF, this condensation operation was repeated once.After washing the resin with DMF and dichloromethane, this was shakenwith 20% piperidine in DMF for 20 minutes to deprotect the Fmoc group,and the resin was washed with DMF to synthesize a protected peptide 74(SEQ ID NO. 81) represented by the following formula (74) on the resin.

After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. Cold diethyl etherwas added to the filtrate, and crude peptide was obtained asprecipitate. This crude peptide was purified with HPLC [column: SHISEIDOCAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluentA: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, A:B=71:29]to obtain glycopeptide 75 (SEQ ID NO. 82) represented by the followingformula (75) (65.7 mg, 11.8 μmol).

ESI-MS: (m/z) calcd for C₂₃₆H₃₅₈N₄₈O₁₀₀S₃: [M+4H]⁴⁺ 1392.0, [M+5H]⁵⁺1113.8, found 1391.9, 1113.8.

31-2 Formation of Disulfide Bond

Glycopeptide 75 obtained in the method described in the above 31-1 (20.3mg, 3.65 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 9.0 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=72:28->67:33, 30 minutes, linear gradient elution] to obtainglycopeptide 76 (SEQ ID NO. 83) represented by the following formula(76) (17.0 mg, 3.06 μmol, yield 84%).

ESI-MS: (m/z) calcd for C₂₃₆H₃₅₆N₄₈O₁₀₀S₃: [M+4H]⁴⁺ 1391.5, [M+5H]⁵⁺1113.4, found 1391.3, 1113.2.

31-3 Deprotection of Benzyl Group

Glycopeptide 76 obtained in the method described in the above 31-2 (7.0mg, 1.3 μmol) was dissolved in 50 mM sodium hydroxide aqueous solution(19.1 mL), and reacted at 0° C. for 1 hour. 200 mM acetic acid aqueoussolution (9.6 mL) was added, and the mixed solution was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10%water/90% acetonitrile, gradient A:B=85:15->77.5:22.5, 20 minutes,linear gradient elution] to obtain a compound represented by thefollowing formula (77) (S1N(disialo)-SRIF28) (SEQ ID NO. 32) (2.7 mg,0.50 μmol, yield 40%)

ESI-MS: (m/z) calcd for C₂₂₂H₃₄₄N₄₈O₁₀₀S₃: [M+3H]³⁺ 1794.9, [M+4H]⁴⁺1346.4, [M+5H]⁵⁺ 1077.3, [M+6H]⁶⁺ 897.9, found 1794.7, 1346.2, 1077.2,897.7.

Example 32 Synthesis of S1C(Disialo).N19C(GlcNAc)-SRIF28 32-1 Synthesisof Peptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF and dichloromethane, in a peptide solid phase synthesis methodwith Fmoc strategy employing a Prelude™ peptide synthesizer, a protectedpeptide was synthesized on a resin. The condensation reaction wasperformed in DMF using HCTU as the condensation agent.

The Fmoc protecting group was removed by treating with 20% piperidine inDMF. After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. A part of the crude peptide waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=72:28->64:36, 20 minutes,linear gradient elution] to obtain peptide 78 (SEQ ID NO. 84)represented by the following formula (78) (28.9 mg).

ESI-MS: (m/z) calcd for C₁₄₆H₂₂₆N₄₂O₃₉S₆: [M+3H]³⁺ 1129.7, [M+4H]⁴⁺847.5, found 1129.5, 847.4.

32-2 Glycosylation of Thiol and Deprotection of StBu Group

Peptide 78 obtained in the method described in the above 32-1 (10.0 mg,2.95 μmol) and compound e represented by the following formula (e)(bromoacetamidated monosaccharide: from Otsuka Chemical Co., Ltd. [0])(2.0 mg, 5.90 μmol, 2.0 equivalents to peptide 78) were dissolved in 33mM phosphate buffer (pH 7.4, 0.89 mL) containing 20 μM TCEP, and reactedat room temperature for 2 hours.

After the reaction, DTT (45.5 mg, 295 μmol) dissolved in 0.1 M phosphatebuffer (pH 7.4, 3.0 mL) was added, and reacted at room temperature for 3hours. The reaction solution was purified with HPLC [column: SHISEIDOCAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluentA: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=75:25->65:35, 20 minutes, linear gradient elution] to obtainglycopeptide 79 (SEQ ID NO. 85) represented by the following formula(79) (6.8 mg, 1.9 μmol, yield 64%).

ESI-MS: (m/z) calcd for C₁₅₂H₂₃₄N₄₄O₄₅S₅[M+3H]³⁺ 1187.0, [M+4H]⁴⁺ 890.5,found 1187.0, 890.5.

32-3 Glycosylation Reaction of Thiol

Peptide 79 obtained in the method described in the above 32-2 (6.8 mg,1.9 μmol) and compound a represented by the above formula (a) (22.4 mg,9.56 μmol, 5.0 equivalents to peptide 79) were dissolved in 0.1 Mphosphate buffer (pH 7.4, 2.0 mL) containing 7.6 mM DTT, and reacted atroom temperature for 2 hours. The reaction solution was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10%water/90% acetonitrile, gradient A:B=85:15->65:35, 20 minutes, lineargradient elution] to obtain glycopeptide 80 (SEQ ID NO. 86) representedby the following formula (80) (3.4 mg, 0.58 μmol, yield 31%).

ESI-MS: (m/z) calcd for C₂₃₈H₃₇₃N₅₁O₁₀₇S₅: [M+5H]⁵⁺ 1165.2, [M+6H]⁶⁺971.2, [M+7H]⁷⁺ 832.6, [M+8H]⁸⁺ 728.6, found 1165.2, 971.1, 832.6,728.6.

32-4 Deprotection of Acm Group

To glycopeptide 80 obtained in the method described in the above 32-3(3.8 mg, 0.65 μmol) was added an aqueous solution (262 μL) of silver(I)acetate (2.7 mg, 16 μmol), and reacted at room temperature for 1 hour.DTT (10.0 mg, 64.8 μmol) dissolved in 100 mM phosphate buffer (pH 7.4,426 μL) and 100 mM ascorbic acid aqueous solution (66 μL) were added,and this was promptly filtered with a filter. The filtrate was purifiedwith HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm,flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10%water/90% acetonitrile, gradient A:B=90:10->60:40, 30 minutes, lineargradient elution] to obtain glycopeptide 81 (SEQ ID NO. 87) representedby the following formula (81) (2.5 mg, 0.44 μmol, yield 67).

ESI-MS: (m/z) calcd for C₂₃₂H₃₆₃N₄₉O₁₀₅S₅: [M+3H]³⁺ 1894.0, [M+4H]⁴⁺1420.7, [M+5H]⁵⁺ 1136.8, found 1893.8, 1420.6, 1136.7.

32-4 Formation of Disulfide Bond

Glycopeptide 81 obtained in the method described in the above 32-3 (2.5mg, 0.44 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 1.1 mL), and reacted overnight at room temperature. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=90:10->70:30, 30 minutes, linear gradient elution] to obtain acompound represented by the following formula (82)(S1C(disialo).N19C(GlcNAc)-SRIF28) (SEQ ID NO. 33) (1.5 mg, 0.26 μmol,yield 59%).

ESI-MS: (m/z) calcd for C₂₃₂H₃₆₁N₄₉O₁₀₅S₅: [M+3H]³⁺ 1893.3, [M+4H]⁴⁺1420.2, [M+5H]⁵ 1136.4, found 1893.5, 1420.1, 1136.3.

Example 33 Synthesis of S1C(Disialo).N19C(diMan)-SRIF28

A compound represented by the following formula (83)(S1C(disialo).N19C(diMan)-SRIF28) (SEQ ID NO. 34) was synthesizedsimilarly to Example 32, except that compound f represented by thefollowing formula (f) (bromoacetamidated oligosaccharide: from OtsukaChemical Co., Ltd.) was employed instead of compound e.

Example 34 Synthesis of C(Disialo)-SRIF28

A compound represented by the following formula (85) (C(disialo)-SRIF28)(SEQ ID NO. 89) was synthesized similarly to Example 1, except that thecompound (peptide 84) (SEQ ID NO. 88) represented by the followingformula (84) was employed instead of peptide 1.

Example 35 Synthesis of R11C(Disialo)-SRIF28

A compound represented by the following formula (87)(R11C(disialo)-SRIF28) (SEQ ID NO. 91) was synthesized similarly toExample 1, except that the compound (peptide 86) (SEQ ID NO. 90)represented by the following formula (86) was employed instead ofpeptide 1.

Example 36 Synthesis of F20C(Disialo)-SRIF28

A compound represented by the following formula (89)(F20C(disialo)-SRIF28) (SEQ ID NO. 93) was synthesized similarly toExample 1, except that the compound (peptide 88) (SEQ ID NO. 92)represented by the following formula (88) was employed instead ofpeptide 1.

Example 37 Synthesis of T24C(Disialo)-SRIF28

A compound represented by the following formula (91)(F20C(disialo)-SRIF28) (SEQ ID NO. 95) was synthesized similarly toExample 1, except that the compound (peptide 90) (SEQ ID NO. 94)represented by the following formula (90) was employed instead ofpeptide 1.

Example 38 Synthesis of F25C(Disialo)-SRIF28

A compound represented by the following formula (93)(F25C(disialo)-SRIF28) (SEQ ID NO. 97) was synthesized similarly toExample 1, except that the compound (peptide 92) (SEQ ID NO. 96)represented by the following formula (92) was employed instead ofpeptide 1.

Example 39 Synthesis of S27C(Disialo)-SRIF28

A compound represented by the following formula (95)(S27C(disialo)-SRIF28) (SEQ ID NO. 99) was synthesized similarly toExample 1, except that the compound (peptide 94) (SEQ ID NO. 98)represented by the following formula (94) was employed instead ofpeptide 1.

Example 40 Synthesis of C(Disialo)-K-SRIF14

A compound represented by the following formula (97)(C(disialo)-K-SRIF14) (SEQ ID NO. 101) was synthesized similarly toExample 1, except that the compound (peptide 96) (SEQ ID NO. 100)represented by the following formula (96) was employed instead ofpeptide 1.

Example 41 Synthesis of S1C(Disialo)-F25Y-SRIF28

A compound represented by the following formula (99)(S1C(disialo)-F25Y-SRIF28) (SEQ ID NO. 103) was synthesized similarly toExample 1, except that the compound (peptide 98) (SEQ ID NO. 102)represented by the following formula (98) was employed instead ofpeptide 1.

Example 42 Synthesis of S1C(Disialo)-SRIF28-Amido

A compound represented by the following formula (101)(S1C(disialo)-SRIF28-amido) (SEQ ID NO. 105) was synthesized similarlyto Example 1, except that the compound (peptide 100) (SEQ ID NO. 104)represented by the following formula (100) was employed instead ofpeptide 1.

Example 43 Synthesis of C(Disialo)-PEGlinker-SRIF14 43-1 Synthesis ofPeptide

The Fmoc protecting group of the protected peptide 39 (SEQ ID NO. 58)(50 μmol) bound to the resin, obtained in the method described in theabove 19-1, was removed by treating with 20% piperidine in DMF. Afterwashing with DMF, employing HCTU as the condensation agent,Fmoc-NH-(PEG)₂-COOH (from Merck) and Fmoc-Cys(Trt)-OH were condensed insequence. The Fmoc protecting group was removed by treating with 20%piperidine in DMF. After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken for 3 hours at room temperature. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. A part of the crude peptide waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=74:26->69:31, 1 minute,then 69:31->62:38, 30 minutes, linear gradient elution] to obtain acompound represented by the following formula (102) (peptide 102) (SEQID NO. 106) (43.1 mg).

ESI-MS: (m/z) calcd for C₉₄H₁₃₈N₂₂O₂₆S₃: [M+2H]²⁺ 1045.2, [M+3H]³⁺697.1, found 1045.0, 697.0.

43-2 Synthesis of C(Disialo)-PEG Linker-SRIF14

A compound represented by the following formula (103) (C(disialo)-PEGlinker-SRIF14) (SEQ ID NO. 107) was synthesized similarly to Example 1,except that peptide 102 obtained in the method described in the above43-1 was employed instead of peptide 1.

Example 44 Synthesis of Biotin-S1C(Disialo)-SRIF28 44-1 Synthesis ofPeptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, adichloromethane (3.0 mL) solution containing Fmoc-Cys(Acm)-OH (49.7 mg,120 μmol) and DIPEA (104.5 μL, 600 μmol) was added, and this was shakenfor 1 hour. After washing with dichloromethane and DMF, the Fmocprotecting group was removed by treating with 20% piperidine in DMF.After washing with DMF, in a peptide solid phase synthesis method withFmoc strategy employing a Prelude™ peptide synthesizer, a protectedpeptide 104 (SEQ ID NO. 108) represented by the following formula (104)was synthesized in a state bound to the resin. The condensation reactionwas performed in DMF using HCTU as the condensation agent.

The Fmoc protecting group was removed by treating with 20% piperidine inDMF. After washing with DMF, employing HCTU as the condensation agent,biotin was condensed. After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. This leads to thedetachment of the protecting group of the amino acid side chain (otherthan the Acm group), as well as cleaving between the peptide and theresin. The resin was filtered off, cold diethyl ether was added to thefiltrate, and crude peptide was obtained as precipitate. The crudepeptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120(5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA,B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=75:25->61:39, 18minutes, linear gradient elution] to obtain peptide 105 (SEQ ID NO. 109)represented by the following formula (105).

ESI-MS: (m/z) calcd for C₁₅₃H₂₃₃N₄₅O₄₂S₅: [M+3H]³⁺ 1179.4, [M+4H]⁴⁺884.8, [M+5H]⁵⁺ 708.0, found 1179.2, 884.4, 707.9.

44-2 Synthesis of Biotin-S1C(Disialo)-SRIF28

A compound represented by the following formula (106)(Biotin-S1C(disialo)-SRIF28) (SEQ ID NO. 110) was synthesized similarlyto Example 1, except that peptide 114 obtained in the method describedin the above 44-1 was employed instead of peptide 1.

Example 45 Synthesis of Biotin-PEG Linker-S1C(Disialo)-SRIF28 45-1Synthesis of Peptide

The Fmoc protecting group of the protected peptide 104 (SEQ ID NO. 108)bound to the resin, obtained in the above 44-1, was removed by treatingwith 20% piperidine in DMF. After washing with DMF, employing HCTU asthe condensation agent, Fmoc-NH-(PEG)₂-COOH (from Merck) and biotin werecondensed in sequence. After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. The crude peptide was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90%acetonitrile, gradient A:B=75:25->61:39, 22 minutes, linear gradientelution] to obtain peptide 107 (SEQ ID NO. 111) represented by thefollowing formula (107).

ESI-MS: (m/z) calcd for C₁₆₂H₂₅₀N₄₆O₄₆S₅: [M+3H]³⁺ 1247.1, [M+4H]⁴⁺935.6, [M+5H]⁵⁺ 748.7, found 1246.9, 935.4, 748.6.

45-2 Synthesis of Biotin-PEG Linker-S1C(Disialo)-SRIF28

A compound represented by the following formula (108) (Biotin-PEGlinker-S1C(disialo)-SRIF28) (SEQ ID NO. 112) was synthesized similarlyto Example 1, except that peptide 107 obtained in the method describedin the above 45-1 was employed instead of peptide 1.

Example 46 Synthesis of Azido-S1C(Disialo)-SRIF28 46-1 Synthesis ofPeptide

The Fmoc protecting group of the protected peptide 104 (SEQ ID NO. 108)bound to the resin, obtained in the above 44-1, was removed by treatingwith 20% piperidine in DMF. After washing with DMF, employing HCTU asthe condensation agent, 5-Azido-pentanoic acid was condensed. Afterwashing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. The crude peptide was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90%acetonitrile, gradient A:B=70:30->60:40, 20 minutes, linear gradientelution] to obtain peptide 109 (SEQ ID NO. 113) represented by thefollowing formula (109).

ESI-MS: (m/z) calcd for C₁₄₈H₂₂₆N₄₆O₄₁S₄: [M+3H]³⁺ 1145.6, [M+4H]⁴⁺859.5, [M+5H]⁵⁺ 687.8, found 1145.5, 859.2, 687.5.

46-2 Synthesis of Azido-S1C(Disialo)-SRIF28

A compound represented by the following formula (110)(Azido-S1C(disialo)-SRIF28) (SEQ ID NO. 114) was synthesized similarlyto Example 1, except that peptide 109 obtained in the method describedin the above 46-1 was employed instead of peptide 1.

Example 47 Synthesis of S1C(Disialo).E12C(Disialo)-SRIF28

A compound represented by the following formula (112)(S1C(disialo).E12C(disialo)-SRIF28) (SEQ ID NO. 116) was synthesizedsimilarly to Example 20, except that a compound represented by thefollowing formula (111) (peptide 111) (SEQ ID NO. 115) was synthesizedand employed instead of peptide 44.

Example 48 Synthesis of 2C(Disialo)-R-K-SRIF14

A compound represented by the following formula (114)(2C(disialo)-R-K-SRIF14) (SEQ ID NO. 118) was synthesized similarly toExample 20, except that a compound represented by the following formula(113) (peptide 113) (SEQ ID NO. 117) was synthesized and employedinstead of peptide 44.

Example 49 Synthesis of 3C(Disialo)-R-K-SRIF14

A compound represented by the following formula (116)(3C(disialo)-R-K-SRIF14) (SEQ ID NO. 120) was synthesized similarly toExample 24, except that a compound represented by the following formula(115) (peptide 115) (SEQ ID NO. 119) was synthesized and employedinstead of peptide 54.

Example 50 Synthesis of S1C(diGlcNAc)-SRIF28 50-1 Glycosylation Reactionof Thiol

Peptide 1 (SEQ ID NO. 38) (from APC, Inc.) (25.0 mg, 7.56 μmol) andcompound h represented by the following formula (h) (bromoacetamidatedoligosaccharide: from Otsuka Chemical Co., Ltd.) (15.6 mg, 15.1 μmol,2.0 equivalents to peptide 1) were dissolved in 33 mM phosphate buffer(pH 7.4, 2.3 mL), and reacted at room temperature for 30 minutes.

The reaction solution was purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, A:B=75:25->62:38,13 minutes, linear gradient elution] to obtain glycopeptide 117 (SEQ IDNO. 121) represented by the following formula (117) (25.6 mg, 6.01 μmol,yield 79%)

ESI-MS: (m/z) calcd for C₁₉₅H₃₀₄N₄₈O₇₆S₄: [M+3H]³⁺ 1556.0, [M+4H]⁴⁺1167.3, found 1555.7, 1167.0.

50-2 Deprotection of Acm Group

To glycopeptide 117 obtained in the method described in the above 50-1(28.3 mg, 6.07 μmol) was added an aqueous solution (2.4 mL) of silver(I)acetate (12.5 mg, 74.5 μmol), and reacted at room temperature for 30minutes. DTT (28.8 mg, 187 μmol) dissolved in 200 mM Tris-HCl buffer (pH7.4, 2.4 mL) and 100 mM ascorbic acid aqueous solution (0.6 mL) wereadded, and this was promptly filtered with a filter. The filtrate waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09%AcOH/10% water/90% acetonitrile, gradient A:B=73:27->60:40, 13 minutes,linear gradient elution] to obtain glycopeptide 118 (SEQ ID NO. 122)represented by the following formula (118) (19.8 mg, 4.38 μmol, yield72%).

ESI-MS: (m/z) calcd for C₁₈₉H₂₉₄N₄₆O₇₄S₄: [M+3H]³⁺ 1508.6, [M+4H]⁴⁺1131.7, [M+5H]⁵ 905.6, found 1508.3, 1131.5, 905.4.

50-3 Formation of Disulfide Bond

Glycopeptide 118 obtained in the method described in the above 50-2(19.8 mg, 4.38 μmol) was dissolved in 100 mM Tris-HCl buffer (pH8.0)-DMSO (1/1, v/v, 8.8 mL), and reacted at room temperature for 2days. The reaction solution was crudely purified with HPLC [column:SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90%acetonitrile, gradient A:B=73:27->60:40, 13 minutes, linear gradientelution] to obtain a fraction containing compound (S1C(diGlcNAc)-SRIF28)represented by the following formula (119) (SEQ ID NO. 123).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=90:10->78:22, 12 minutes, linear gradient elution] to obtainS1C(diGlcNAc)-SRIF28 (11.9 mg, 2.63 μmol, yield 60%).

ESI-MS: (m/z) calcd for C₁₈₉H₂₉₂N₄₆O₇₄S₄: [M+3H]³⁺ 1508.0, [M+4H]⁴⁺1131.2, [M+5H]⁵⁺ 905.2, found 1507.7, 1131.0, 905.0.

Example 51 Synthesis of S1C(diMan)-SRIF28

A compound represented by the following formula (120)(S1C(diMan)-SRIF28) (SEQ ID NO. 124) was synthesized similarly toExample 50, except that compound f was employed instead of compound h.

Example 52 Synthesis of N19C(diMan)-SRIF28

A compound represented by the following formula (121)(N19C(diMan)-SRIF28) (SEQ ID NO. 125) was synthesized similarly toExample 50, except that peptide 21 was employed instead of peptide 1 andcompound f was employed instead of compound h.

Example 53 Synthesis of S1C(GlcNAc)-SRIF28

A compound represented by the following formula (122) (S1C(GlcNAc)-SRIF28) (SEQ ID NO. 126) was synthesized similarly to Example50, except that compound e was employed instead of compound h.

Example 54 Synthesis of N19C(GlcNAc)-SRIF28

A compound represented by the following formula (123) (N19C(GlcNAc)-SRIF28) (SEQ ID NO. 127) was synthesized similarly to Example50, except that peptide 21 was employed instead of peptide 1 andcompound e was employed instead of compound h.

Example 55 Synthesis of S1C(Trisialo)-SRIF28

A compound represented by the following formula (124)(S1C(trisialo)-SRIF28) (SEQ ID NO. 128) was synthesized similarly toExample 1, except that compound i represented by the following formula(i) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.)was employed instead of compound a.

Example 56 Synthesis of S1C(Tetrasialo)-SRIF28

A compound represented by the following formula (125)(S1C(tetrasialo)-SRIF28) (SEQ ID NO. 129) was synthesized similarly toExample 1, except that compound j represented by the following formula(j) (bromoacetamidated oligosaccharide: from Otsuka Chemical Co., Ltd.)was employed instead of compound a.

Example 57 Synthesis of S1C(Disialo(Aminoethylamide))-SRIF28 57-1Synthesis of Bromoacetamidated Disialo Sugar Chain Derivative

To compound k represented by the following formula (k) (from OtsukaChemical Co., Ltd.) (204.1 mg, 92.1 μmol) were added water (2 mL) andtert-butyl N-(2-aminoethyl)carbamate (0.29 mL, 0.18 mmol), and this wasstirred at room temperature for 1 hour. After lyophilization, to thelyophilizate obtained were added DMF (5 mL), HATU (349 mg, 921 μmol),and DIPEA (161 μL, 921 μmol), and reacted at 37° C. for 18 hours.Toluene (50 mL) was added to the solution, and the deposited precipitatewas collected by filtration. The precipitate was dissolved in DMF (5mL), purified with gel filtration purification [column: Sephadex G-25,φ20×200 mm, flow rate: 30 mL/h], and the fraction of interest wascollected and lyophilized to obtain compound 1 represented by thefollowing formula (1) (152.4 mg, 60.8 μmol, yield 66%).

MALDI-MS: (m/z) calcd for C₉₈H₁₆₆N₁₀O₆₄: [M+Na]⁺ 2530.0, found 2529.4.

Compound 1 (100 mg, 39.8 μmol) and ammonium hydrogen carbonate (31.4 mg,398 μmol) were dissolved in water (1 mL), and reacted at roomtemperature for 7 days. After lyophilization, to the lyophilizateobtained were sequentially added water (1 mL), DCC (41.0 mg, 199 μmol),and bromoacetic acid (27.7 mg, 199 μmol) dissolved in DMF (1 mL). After1 hour of reaction under ice cooling, the solution was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 8.0 mL/min, eluent:water:acetonitrile=84:16] to obtain compound mrepresented by the following formula (m) (bromoacetamidated disialosugar chain derivative: 100 mg, 38.2 μmol, yield 96%).

MALDI-MS: (m/z) (m/z) calcd for C₁₀₀H₁₆₈BrN₁₁O₆₄: [M+Na]⁺ 2648.9, found2648.5.

57-2 Glycosylation Reaction of Thiol

Compound m obtained in the method described in the above 57-1 (14.2 mg,5.40 μmol) and peptide 1 (15.0 mg, 4.53) μmol were dissolved in 33 mMphosphate buffer (pH 7.4, 1.3 mL), and reacted at room temperature for30 minutes. The reaction solution was purified with HPLC [column:SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0mL/min, eluent A: 0.1% acetic acid (AcOH) water, B: 0.09% AcOH/10%water/90% acetonitrile, gradient A:B=86:14->82:18, 20 minutes, lineargradient elution] to obtain glycopeptide 126 (SEQ ID NO. 130)represented by the following formula (126) (13.4 mg, 2.29 μmol, yield51%).

ESI-MS: (m/z) calcd for C₂₄₃H₃₈₆N₅₄O₁₀₄S₄: [M+4H]⁴⁺ 1465.1, [M+5H]⁵⁺1172.2, [M+6H]⁶⁺ 977.0, found 1464.9, 1172.1, 977.1.

57-3 Deprotection of Boc Group

Glycopeptide 126 obtained in the method described in the above 57-2(13.4 mg, 2.29 μmol) was dissolved in 95% TFA aqueous solution (458 μL),and this was shaken at room temperature for 5 minutes. After adding 50mM ammonium acetate aqueous solution (pH 6.8, 33 mL), the reactionsolution was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120(5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA,B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27->65:35, 10minutes, linear gradient elution] to obtain glycopeptide 127 (SEQ ID NO.131) represented by the following formula (127) (12.7 mg, 98 μmol, yield98%).

ESI-MS: (m/z) calcd for C₂₃₃H₃₇₀N₅₄O₁₀₀S₄: [M+4H]⁴⁺ 1415.1, [M+5H]⁵⁺1132.2, [M+6H]⁶⁺ 943.7, [M+7H]⁷⁺ 809.0, found 1414.9, 1132.1, 943.6,808.9.

57-4 Deprotection of Acm Group

To glycopeptide 127 obtained in the method described in the above 57-3(12.7 mg, 2.25 μmol) was added an aqueous solution (0.9 mL) of silver(I)acetate (9.2 mg, 55 μmol), and reacted at room temperature for 30minutes. Then, DTT (21.2 mg, 137 μmol) dissolved in 200 mM phosphatebuffer (pH 7.4, 0.9 mL) and 100 mM ascorbic acid aqueous solution (225μL) were added, and this was promptly filtered with a filter. Thefiltrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120(5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA,B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27->60:40, 13minutes, linear gradient elution] to obtain glycopeptide 128 (SEQ ID NO.132) represented by the following formula (128) (5.2 mg, 0.94 μmol,yield 42%).

ESI-MS: (m/z) calcd for C₂₂₇H₃₆₀N₅₂O₉₈S₄: [M+4H]⁴⁺ 1379.5, [M+5H]⁵⁺1103.8, [M+6H]⁶⁺ 920.0, [M+7H]⁷⁺ 788.7, found 1379.4, 1103.7, 919.9,788.6.

57-5 Formation of Disulfide Bond

Glycopeptide 128 obtained in the method described in the above 57-4 (5.2mg, 0.94 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 1.9 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=70:30->65:35, 13 minutes, linear gradient elution] to obtain afraction containing compound (S1C(disialo(aminoethylamide))-SRIF28)represented by the following formula (129) (SEQ ID NO. 133).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=92:8->85:15, 14 minutes, linear gradient elution] to obtainS1C(disialo(aminoethylamide))-SRIF28 (3.8 mg, 0.69 μmol, yield 73%).

ESI-MS: (m/z) calcd for C₂₂₇H₃₅₈N₅₂O₉₈S₄: [M+3H]³⁺ 1838.3, [M+4H]⁴⁺1379.0, [M+5H]⁵⁺ 1103.4, [M+6H]⁶⁺ 919.6, [M+7H]⁷⁺ 788.4, found 1838.0,1378.5, 1103.2, 919.5, 788.2.

Example 58 Synthesis of S1C(Disialo(Amide))-SRIF28 58-1 Synthesis ofBromoacetamidated Disialo Sugar Chain Derivative

To compound k (from Otsuka Chemical Co., Ltd.) (152 mg, 68.6 μmol) weresequentially added DMF (1.9 mL), lithium bromide (357 mg, 4.12 mmol),and phenacyl chloride (273 mg, 1.37 mmol), and reacted at 37° C. After10 hours, water (19 mL) was added and the precipitate was removed byfiltration. To the filtrate was added 25% ammonium water (5 mL), andreacted at room temperature for 18 hours, and then 100 mM phosphatebuffer (pH 7.4, 80 mL) was added to allow neutralization. The solutionwas purified with HPLC [column: YMC Hydrosphere C18 (5 μm), φ20×250 mm,flow rate: 8.0 mL/min, eluent: 0.1% aqueous TFA:acetonitrile=98:2->92:8,30 minutes, linear gradient elution] and lyophilized to obtain compoundn represented by the following formula (n) (60.9 mg, 27.4 μmol, yield40%).

MALDI-MS: (m/z) calcd for C₈₄H₁₄₀N₈O₆₀: [M+Na]⁺ 2243.8, found 2243.6.

The obtained intermediate n (37.3 mg, 16.8 μmol) and ammonium hydrogencarbonate (13.3 mg, 168 μmol) were dissolved in water (0.37 mL), andreacted at room temperature for 7 days. After lyophilization, to the [0]lyophilizate obtained were sequentially added water (0.37 mL), DCC (17.3mg, 84 μmol), and bromoacetic acid (11.7 mg, 84 μmol) dissolved in DMF(0.37 mL). The solution after 1 hour of reaction under ice cooling waspurified with HPLC [column: YMC Hydrosphere C18 (5 μm), φ20×250 mm, flowrate: 8.0 mL/min, eluent: 0.1% aqueous TFA:acetonitrile=98:2->92:8, 30minutes, linear gradient elution] to obtain compound o represented bythe following formula (o) (bromoacetamidated disialo sugar chainderivative: 29.0 mg, 12.4 μmol, yield 74%).

MALDI-MS: (m/z) calcd for C₈₆H₁₄₂BrN₉O₆₀: [M+Na]⁺ 2362.7, found 2362.5.

58-2 Synthesis of S1C(Disialo(Amide))-SRIF28

A compound represented by the following formula (130)(S1C(disialo(amide))-SRIF28) (SEQ ID NO. 134) was synthesized similarlyto Example 1, except that compound o obtained in the method described inthe above 58-1 was employed instead of compound a.

Example 59 Synthesis of S1C(Disialo (Bn))-SRIF28 59-1 Synthesis ofDisialo Sugar Chain Having Bromoacetamidated Benzyl Protecting Group

To compound a (28.9 mg, 12.3 μmol) were sequentially added DMF (0.58mL), lithium bromide (21.5 mg, 248 μmol), and benzyl bromide (14.6 μL,122 μmol), and reacted at 30° C. for 20 hours. Benzyl bromide (14.6 μL,122 μmol) was further added and reacted for 20 hours. To the reactionsolution was added toluene (30 mL), and after centrifugal separation(10,000×g, 10 minutes), the precipitate was dissolved in water (100 μL)and purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 8.0 mL/min,eluent:water:acetonitrile=95:5->70:30, 20 minutes, linear gradientelution] to obtain compound g represented by the following formula (g)(bromoacetamidated disialo sugar chain: 7.6 mg, 3.0 μmol, yield 24%).

MALDI-MS: (m/z) calcd for C₁₀₀H₁₅₂BrN₇O₆₂: [M+Na]⁺ 2544.8, found 2544.4.

Example 59-2 Synthesis of S1C(Disialo(Bn))-SRIF28

A compound represented by the following formula (131)(S1C(disialo(Bn))-SRIF28) (SEQ ID NO. 135) was synthesized similarly toExample 1, except that compound g was employed instead of compound a.

Example 60 Synthesis of S1C(Disialo (Hexadecylamide))-SRIF28 60-1Synthesis of Bromoacetamidated Disialo Sugar Chain Derivative

To compound k (from Otsuka Chemical Co., Ltd.) (140 mg, 63.0 μmol) wereadded water (1.5 mL), methanol (1.5 mL), and hexadecylamine (300 mg, 126μmol), and this was stirred at room temperature for 1 hour. Afterlyophilization, to the lyophilizate obtained were added DMF (5 mL), HATU(239 mg, 630 μmol), and DIPEA (110 μL, 630 μmol), and reacted at 37° C.After 18 hours, diethyl ether (100 mL) was added to the solution, andthe deposited precipitate was collected by filtration. This precipitatewas dissolved in DMF (5 mL) and purified with HPLC [column: SHISEIDOCAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 8.0 mL/min,eluent:water:acetonitrile=40:60->10:90, 30 minutes, linear gradientelution] to obtain compound p represented by the following formula (p)(71.1 mg, 26.6 μmol, yield 42%).

MALDI-MS: (m/z) calcd for C₁₁₆H₂₀₄N₈O₆₀: [M+Na]⁺ 2692.3, found 2691.9.

The obtained compound p (71.7 mg, 26.6 μmol) and ammonium hydrogencarbonate (21.8 mg, 266 μmol) were dissolved in water (0.7 mL) andmethanol (0.7 mL), and reacted at room temperature. After 7 days, to thelyophilizate obtained by lyophilization were sequentially added water(0.7 mL), methanol (0.7 mL), DCC (27.4 mg, 133 μmol), and bromoaceticacid (18.5 mg, 133 μmol) dissolved in DMF (0.7 mL). After 1 hour ofreaction under ice cooling, the solution was purified with HPLC [column:SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 8.0mL/min, eluent:water:acetonitrile=40:60->10:90, 30 minutes, lineargradient elution] to obtain compound q represented by the followingformula (q) (bromoacetamidated disialo sugar chain: 24.9 mg, 8.9 μmol,yield 33%).

MALDI-MS: (m/z) calcd for C₁₁₈H₂₀₆BrN₉O₆₀: [M+Na]⁺ 2811.2, found 2811.0.

60-2 Glycosylation Reaction of Thiol

Peptide 1 (10.4 mg, 3.14 μmol) was dissolved in 0.5 M phosphate buffer(pH 7.4, 62 μL) containing 30 μM TCEP. To this solution was added asolution of compound q obtained in the method described in the above60-1 (79.4 mg, 28.5 μmol) in DMSO (3.7 mL), and reacted at roomtemperature for 20 minutes. The reaction solution was purified with HPLC[column: SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min,eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile,gradient A:B=50:50->22:78, 14 minutes, linear gradient elution] toobtain glycopeptide 132 (SEQ ID NO. 136) represented by the followingformula (132) (6.4 mg, 1.1 μmol, yield 35%).

ESI-MS: (m/z) calcd for C₂₆₁H₄₂₄N₅₂O₁₀₀S₄: [M+3H]³⁺ 2007.2, [M+4H]⁴⁺1505.7, [M+5H]⁵⁺ 1204.7, [M+6H]⁶⁺ 1004.1, found 2007.4, 1505.5, 1204.8,1004.0.

60-3 Deprotection of Acm Group

To glycopeptide 132 obtained in the method described in the above 60-2(6.4 mg, 1.1 μmol) was added an aqueous solution (0.8 mL) of silver(I)acetate (3.8 mg, 23 μmol), and reacted at room temperature for 40minutes. Then, DTT (8.8 mg, 57 μmol) dissolved in 200 mM phosphatebuffer (pH 7.4, 377 μL) and 100 mM ascorbic acid aqueous solution (106μL) were added, and this was promptly filtered with a filter. Thefiltrate was purified with HPLC [column: SHISEIDO Proteonavi (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, gradient A:B=48:52->38:62, 3 minutes,then 38:62->30:70, 8 minutes, linear gradient elution] to obtainglycopeptide 133 (SEQ ID NO. 137) represented by the following formula(133) (3.2 mg, 0.54 μmol, yield 49%).

ESI-MS: (m/z) calcd for C₂₅₅H₄₁₄N₅₀O₉₈S₄: [M+3H]³⁺ 1959.9, [M+4H]⁴⁺1470.1, [M+5H]⁵⁺ 1176.3, [M+6H]⁶⁺ 980.4, found 1959.6, 1469.9, 1176.1,980.5.

60-3 Formation of Disulfide Bond

Glycopeptide 133 obtained in the method described in the above 60-2 (3.2mg, 0.54 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 1.1 mL), and reacted at room temperature for 2 days. Thereaction solution was purified with HPLC [column: SHISEIDO Proteonavi (5μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B:0.09% TFA/10% water/90% acetonitrile, gradient A:B=48:52->38:62, 3minutes, then 38:62->30:70, 8 minutes, linear gradient elution] toobtain a compound represented by the following formula (134)(S1C(disialo (hexadecylamide))-SRIF28) (SEQ ID NO. 138) (2.8 mg, 0.48μmol, yield 89%).

ESI-MS: (m/z) calcd for C₂₅₅H₄₁₂N₅₀O₉₈S₄: [M+3H]³⁺ 1959.2, [M+4H]⁴⁺1469.6, [M+5H]⁵⁺ 1175.9, [M+6H]⁶⁺ 980.1, found 1958.9, 1469.4, 1175.7,979.9.

Example 61 Synthesis of S1-2C(Disialo(Amide))-SRIF28

A compound represented by the following formula (135)(S1-2C(disialo(amide))-SRIF28) (SEQ ID NO. 139) was synthesizedsimilarly to Example 28, except that compound o was employed instead ofcompound c.

Example 62 Synthesis of S1-2C(Disialo(Bn))-SRIF28

A compound represented by the following formula (136)(S1-2C(disialo(Bn))-SRIF28) (SEQ ID NO. 140) was synthesized similarlyto Example 28, except that compound g was employed instead of compoundc.

Example 63 Synthesis of S1C(Asn(Disialo))-SRIF28

A compound represented by the following formula (137)(S1C(Asn(disialo))-SRIF28) (SEQ ID NO. 141) was synthesized similarly toExample 1, except that compound r represented by the following formula(r) (bromoacetylated glycosylated Asn: from Otsuka Chemical Co., Ltd.)was employed instead of compound a.

Example 64 Synthesis of S1N(Disialo).N19C(diMan)-SRIF28 64-1 Solid PhaseSynthesis of Glycopeptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Trt)-OH (72.5 mg, 120 μmol) and DIPEA (104.6 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF, in a peptide solid phase synthesis method with Fmoc strategyemploying a Prelude™ peptide synthesizer, a protected peptide 138 (SEQID NO. 142) represented by the following formula (138) was synthesizedin a state bound to the resin. The condensation reaction was preformedin DMF using HCTU as the condensation agent.

Next, the Fmoc protecting group was removed by treating with 20%piperidine in DMF. After washing with DMF, compound d (141.0 mg, 51.5μmol), DMSO-DMF (1/1, v/v, 1.1 mL) solution, TBTU (21.2 mg, 66.0 μmol),and DIPEA (17.2 μL, 98.7 μmol) were sequentially added to the resin, andthis was shaken at room temperature for 4 hours to allow condensation.After washing with DMF, this condensation operation was repeated once.After washing the resin with DMF and dichloromethane, this was shakenwith 20% piperidine in DMF for 20 minutes to deprotect the Fmoc group,and the resin was washed with DMF to synthesize a protected peptide 139(SEQ ID NO. 143) represented by the following formula (139) in a statebound to the resin.

After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. This crude peptide was purifiedwith HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm,flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10%water/90% acetonitrile, A:B=70:30] to obtain glycopeptide 140 (SEQ IDNO. 144) represented by the following formula (140) (5.8 mg, 1.1 μmol).

64-2 Glycosylation Reaction of Thiol

Glycopeptide 140 obtained in the method described in the above 64-1(10.8 mg, 1.90 μmol) and compound f (5.3 mg, 5.1 μmol) were dissolved in100 mM phosphate buffer (pH 7.4, 0.8 mL) containing 141 μM TCEP, andreacted at room temperature for 24 hours. The reaction solution waspurified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09%TFA/10% water/90% acetonitrile, A:B=75:25->60:40, 30 minutes, lineargradient elution] to obtain glycopeptide 141 (SEQ ID NO. 145)represented by the following formula (141) (4.9 mg, 0.74 μmol, yield39%).

ESI-MS: (m/z) calcd for C₂₇₇H₄₂₆N₅₂O₁₂₇S₄: [M+3H]³⁺ 2216.0, [M+4H]⁴⁺1662.2, [M+5H]⁵⁺ 1330.0, found 2215.6, 1661.9, 1330.1.

64-3 Deprotection of Acm Group

To glycopeptide 141 obtained in the method described in the above 64-2(4.9 mg, 0.74 μmol) was added an aqueous solution (148 μL) of silver(I)acetate (1.5 mg, 9.0 μmol), and reacted at room temperature for 1.5hours reaction. DTT (3.5 mg, 23 μmol) dissolved in 200 mM phosphatebuffer (pH 7.4, 145 μL) and 100 mM ascorbic acid aqueous solution (37μL) were added, and this was promptly filtered with a filter. Thefiltrate was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120(5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA,B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=74:26->64:36, 30minutes, linear gradient elution] to obtain glycopeptide 142 (SEQ ID NO.146) represented by the following formula (142) (3.7 mg, 0.57 μmol,yield 77%)

ESI-MS: (m/z) calcd for C₂₇₁H₄₁₆N₅₀O₂₅S₄: [M+3H]³⁺ 2168.6, [M+4H]⁴⁺1626.7, [M+5H]⁵⁺ 1301.5, found 2168.6, 1626.4, 1301.3.

64-4 Formation of Disulfide Bond

Glycopeptide 142 obtained in the method described in the above 64-3 (3.7mg, 0.54 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 1.4 mL), and reacted at room temperature for 31 hours. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=75:25->60:40, 30 minutes, linear gradient elution] to obtainglycopeptide 143 (SEQ ID NO. 147) represented by the following formula(143) (2.9 mg, 0.45 μmol, yield 78%).

ESI-MS: (m/z) calcd for C₂₇₁H₄₁₄N₅₀O₁₂₅S₄: [M+3H]³⁺ 2167.9, [M+4H]⁴⁺1626.2, [M+5H]⁵⁺ 1301.1, found 2167.9, 1626.0, 1301.2.

64-5 Deprotection of Benzyl Group

Glycopeptide 143 obtained in the method described in the above 64-4 (2.9mg, 0.45 μmol) was dissolved in 50 mM sodium hydroxide aqueous solution(13.6 mL), and reacted at 0° C. for 1 hour. 200 mM acetic acid aqueoussolution (3.4 mL) was added, and the mixed solution was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 7.0 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90%acetonitrile, gradient A:B=75:25->60:40, 20 minutes, linear gradientelution] to obtain a fraction containing compound(S1N(disialo).N19C(diMan)-SRIF28) represented by the following formula144 (SEQ ID NO. 148).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ4.6×250 mm, flow rate: 0.7 mL/min, eluent A:0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=95:5->85:15, 2 minutes, then 85:15->65:35, 20 minutes, lineargradient elution] to obtain S1N(disialo).N19C(diMan)-SRIF28 (1.6 mg,0.25 μmol, yield 57%).

ESI-MS: calcd for C₂₅₇H₄₀₂N₅₀O₁₂₅S₄: [M+3H]³⁺ 2107.8, [M+4H]⁴⁺ 1581.1,[M+5H]⁵⁺ 1265.1, found 2107.9, 1580.9, 1265.1.

Example 65 Synthesis of C(Disialo(Aminoethylamide)).S1C(Disialo)-SRIF2865-1 Synthesis of Peptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF, in a peptide solid phase synthesis method with Fmoc strategyemploying a Prelude™ peptide synthesizer, a protected peptide 145 (SEQID NO. 149) represented by the following formula (145) was synthesizedin a state bound to the resin. The condensation reaction was performedin DMF using HCTU as the condensation agent.

A part of the resin (50 μmol) was taken, and the Fmoc protecting groupwas removed by treating with 20% piperidine in DMF. After washing withDMF and dichloromethane, TFA:water:triisopropylsilane:ethanedithiol(=90:2.5:5:2.5) was added, and this was shaken at room temperature for 3hours. The resin was filtered off, cold diethyl ether was added to thefiltrate, and crude peptide was obtained as precipitate. The crudepeptide was purified with HPLC [column: SHISEIDO CAPCELL PAK C18 UG-120(5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous TFA,B: 0.09% TFA/10% water/90% acetonitrile, gradient A:B=73:27->63:37, 30minutes, linear gradient elution] to obtain peptide 146 (SEQ ID NO. 150)represented by the following formula (146) (30.4 mg).

ESI-MS: (m/z) (m/z) calcd for C₁₅₀H₂₃₂N₄₄O₄₁S₆: [M+3H]³⁺ 1167.7,[M+4H]⁴⁺ 876.0, found 1167.5, 875.9.

65-2 Deprotection of Boc Group in Sugar Chain Derivative

Compound m (50.0 mg, 19.0 μmol) was dissolved in a 95% aqueous TFA (2.5mL), and this was shaken at room temperature. After 10 minutes, diethylether (15 mL) was added, and the deposited precipitate was centrifuged(10,000×g 10 minutes). The precipitate was dissolved in water andlyophilized to obtain compound s represented by the following formula(s) (bromoacetamidated disialo sugar chain: 46.0 mg, 18.9 μmol, yield99%).

ESI-MS: (m/z) calcd for C₉₀H₁₅₂BrN₁₁O₆₀: [M+2H]²⁺ 1215.1, [M+3H]³⁺810.4, found 1214.9, 810.3.

65-3 Glycosylation of Thiol

Peptide 146 obtained in the method described in the above 65-1 (20.6 mg,5.89 μmol) and compound s obtained in the method described in the above65-2 (28.6 mg, 11.8 μmol) were dissolved in 33 mM phosphate buffer (pH7.4, 1.8 mL), and reacted at room temperature for 30 minutes. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=74:26->64:36, 20 minutes, linear gradient elution] to obtainglycopeptide 147 (SEQ ID NO. 151) represented by the following formula(147) (17.1 mg, 2.93 μmol, yield 50%).

ESI-MS: (m/z) calcd for C₂₄₀H₃₈₃N₅₅O₁₀₁S₆: [M+3H]³⁺ 1950.1, [M+4H]⁴⁺1462.8, [M+5H]⁵⁺ 1170.5, [M+6H]⁶⁺ 975.6, found 1949.9, 1462.6, 1170.3,975.4.

65-4 Deprotection of StBu Group

To glycopeptide 147 obtained in the method described in the above 65-3(17.1 mg, 2.93 μmol) was added DTT (52.9 mg, 343 μmol) dissolved in 0.1M phosphate buffer (pH 7.4, 3.4 mL), and reacted at room temperature for3 hours. The reaction solution was purified with HPLC [column: SHISEIDOCAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluentA: 0.1% aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=95:5->75:25, 20 minutes, linear gradient elution] to obtainglycopeptide 148 (SEQ ID NO. 152) represented by the following formula(148) (8.6 mg, 1.5 μmol, yield 51%).

ESI-MS: (m/z) calcd for C₂₃₆H₃₇₅N₅₅O₁₀₁S₅: [M+3H]³⁺ 1920.7, [M+4H]⁴⁺1440.8, [M+5H]⁵⁺ 1152.8, [M+6H]⁶⁺ 960.9, [M+7H]⁷⁺ 823.7, found 1920.5,1440.6, 1152.7, 960.6, 823.6.

65-5 Glycosylation Reaction of Thiol

Peptide 148 obtained in the method described in the above 65-4 (6.2 mg,1.1 μmol) and compound a (3.8 mg, 1.6 μmol) were dissolved in 0.36 Mphosphate buffer (pH 7.4, 339 mL) containing 1.6 mM DTT, and reacted atroom temperature for 2.5 hours The reaction solution was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10%water/90% acetonitrile, gradient A:B=90:10->75:25, 30 minutes, lineargradient elution] to obtain glycopeptide 149 (SEQ ID NO. 153)represented by the following formula (149) (6.4 mg, 0.80 μmol, yield73%).

ESI-MS: (m/z) calcd for C₃₂₂H₅₁₄N₆₂O₁₆₃S₅: [M+4H]⁴⁺ 2006.5, [M+5H]⁵⁺1605.4, [M+6H]⁶⁺ 1338.0, found 2006.6, 1605.3, 1338.0.

65-6 Deprotection of Acm Group

To glycopeptide 149 obtained in the method described in the above 65-5(8.2 mg, 1.0 μmol) was added an aqueous solution (225 μL) of silver(I)acetate (2.1 mg, 13 μmol), and reacted at room temperature for 1 hour.DTT (4.8 mg, 31 μmol) dissolved in 100 mM phosphate buffer (pH 7.4, 204μL) and 100 mM ascorbic acid aqueous solution (51 μL) were added, andthis was promptly filtered with a filter. The filtrate was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ20×250 mm, flowrate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09% AcOH/10%water/90% acetonitrile, gradient A:B=90:10->75:25, 30 minutes, lineargradient elution] to obtain glycopeptide 150 (SEQ ID NO. 154)represented by the following formula (150) (5.5 mg, 0.70 μmol, yield70%).

ESI-MS: (m/z) calcd for C₃₁₆H₅₀₄N₆₀O₁₆₁S₅: [M+4H]⁴⁺ 1971.0, [M+5H]⁵⁺1577.0, [M+6H]⁶⁺ 1314.3, found 1970.6, 1576.8, 1314.2.

65-7 Formation of Disulfide Bond

Glycopeptide 150 obtained in the method described in the above 65-6 (5.4mg, 0.69 μmol) was dissolved in 100 mM Tris-HCl buffer (pH 8.0)-DMSO(1/1, v/v, 1.7 mL), and reacted overnight at room temperature. Thereaction solution was purified with HPLC [column: SHISEIDO CAPCELL PAKC18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous TFA, B: 0.09% TFA/10% water/90% acetonitrile, gradientA:B=75:25->65:35, 30 minutes, linear gradient elution] to obtain afraction containing compound(C(disialo(aminoethylamide))/S1C(disialo)-SRIF28) represented by thefollowing formula 151 (SEQ ID NO. 155).

This fraction was further purified with HPLC [column: SHISEIDO CAPCELLPAK C18 UG-120 (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1%aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=90:10->75:25, minutes, linear gradient elution] to obtainC(disialo(aminoethylamide))/S1C(disialo)-SRIF28 (3.1 mg, 0.40 μmol,yield 58%).

ESI-MS: calcd for C₃₁₆H₅₀₂N₆₀O₁₆₁S₅: [M+4H]⁴⁺ 1970.5, [M+5H]⁵⁺ 1576.6,[M+6H]⁶⁺ 1314.0, [M+7H]⁷⁺ 1126.4, [M+8H]⁸⁺ 985.8, [M+9H]⁹⁺ 876.3, found1970.3, 1576.4, 1313.9, 1126.4, 985.5, 876.1.

Example 66 Synthesis of S1-4C(Disialo)-SRIF28 66-1 Synthesis of Peptide

2-chlorotrityl chloride resin (100 μmol) was taken in a column for solidphase synthesis, and after washing with DMF and dichloromethane, asolution of Fmoc-Cys(Acm)-OH (49.7 mg, 120 μmol) and DIPEA (104.5 μL,600 μmol) in dichloromethane (3.0 mL) was added, and this was shaken for1 hour. After washing with dichloromethane and DMF, the Fmoc protectinggroup was removed by treating with 20% piperidine in DMF. After washingwith DMF, in a peptide solid phase synthesis method with Fmoc strategyemploying a Prelude™ peptide synthesizer, a protected peptide 152 (SEQID NO. 156) represented by the following formula (152) was synthesizedin a state bound to the resin. The condensation reaction was performedin DMF using HCTU as the condensation agent.

The Fmoc protecting group was removed by treating with 20% piperidine inDMF. After washing with DMF and dichloromethane,TFA:water:triisopropylsilane:ethanedithiol (=90:2.5:5:2.5) was added,and this was shaken at room temperature for 3 hours. The resin wasfiltered off, cold diethyl ether was added to the filtrate, and crudepeptide was obtained as precipitate. The crude peptide was purified withHPLC [column: SHISEIDO CAPCELL PAK C18 UG-120 (5 μm), φ50×250 mm, flowrate: 43.7 mL/min, eluent A: 0.1% aqueous TFA, B: 0.09% TFA/10%water/90% acetonitrile, gradient A:B=75:25->65:35, 20 minutes, lineargradient elution] to obtain peptide 153 (SEQ ID NO. 157) represented bythe following formula (153) (127.2 mg).

ESI-MS: (m/z) calcd for C₁₅₂H₂₃₄N₄₆O₄₃S₇: [M+3H]³⁺ 1207.1, [M+4H]⁴⁺905.6, [M+5H]⁵⁺ 724.6, found 1206.9, 905.1, 724.5.

66-2 Glycosylation Reaction of Thiol

Peptide 153 obtained in the method described in the above 66-1 (30.4 mg,8.40 μmol) and compound a (128 mg, 54.7 μmol) were dissolved in 33 mMphosphate buffer (pH 7.4, 2.5 mL), and reacted overnight at roomtemperature. The reaction solution was purified with HPLC [column:SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A:0.1% aqueous AcOH, B: 0.09% AcOH/10% water/90% acetonitrile, gradientA:B=82:18->71:29, 20 minutes, linear gradient elution] to obtainglycopeptide 154 (SEQ ID NO. 158) represented by the following formula(154) (30.9 mg, 2.44 μmol, yield 29%).

ESI-MS: (m/z) calcd for C₄₉₆H₇₉₀N₇₄O₂₉₁S₇: [M+6H]⁶⁺ 2112.7, [M+7H]⁷⁺1811.1, found 2112.8, 1811.0.

66-3 Deprotection of Acm Group

To glycopeptide 154 obtained in the method described in the above 66-2(30.9 mg, 2.44 μmol) was added an aqueous solution (0.98 mL) ofsilver(I) acetate (5.0 mg, 30 μmol), and reacted at room temperature for20 minutes. Then, DTT (11.8 mg, 76.5 μmol) dissolved in 200 mM Tris-HClbuffer (pH 7.4, 0.98 mL) and 100 mM ascorbic acid aqueous solution (244μL) were added, and this was promptly filtered with a filter. Thefiltrate was purified with HPLC [column: SHISEIDO Proteonavi (5 μm),φ20×250 mm, flow rate: 7.0 mL/min, eluent A: 0.1% aqueous AcOH, B: 0.09%AcOH/10% water/90% acetonitrile, gradient A:B=82:18->70:30, 20 minutes,linear gradient elution] to obtain glycopeptide 155 (SEQ ID NO. 159)represented by the following formula (155) (20.6 mg, 1.64 μmol, yield67%).

ESI-MS: (m/z) calcd for C₄₉₀H₇₈₀N₇₂O₂₈₉S₇: [M+5H]⁵⁺ 2506.6, [M+6H]⁶⁺2089.0, [M+7H]⁷⁺ 1790.7, found 2506.5, 2088.8, 1790.4.

66-4 Formation of Disulfide Bond

Glycopeptide 155 obtained in the method described in the above 66-3(20.6 mg, 1.64 μmol) was dissolved in 100 mM Tris-HCl buffer (pH8.0)-DMSO (1/1, v/v, 2.1 mL), and reacted at room temperature for 2days. Then, the reaction solution was purified with HPLC [column:SHISEIDO Proteonavi (5 μm), φ20×250 mm, flow rate: 7.0 mL/min, eluent A:10 mM ammonium acetate aqueous solution, B: 10 mM ammoniumacetate-acetonitrile (1/9, v/v), gradient A:B=75:25->72:28, 15 minutes,linear gradient elution] to obtain S1-4C(disialo)-SRIF28 (SEQ ID NO.160) represented by the following formula (156) (1116 mg, 0.93 μmol,yield 57%).

ESI-MS: (m/z) calcd for C₄₉₀H₇₇₈N₇₂O₂₈₉S₇: [M+5H]⁵⁺ 2506.2, [M+6H]⁶⁺2088.7, [M+7H]⁷⁺ 1790.5, found 2506.1, 2088.6, 1790.4.

Tables 1-1 to 1-7 show the MS spectrum data (ESI-MS) of the glycosylatedSRIF peptides obtained in the methods described in Examples 1-66. Themolecular mass was obtained by performing deconvolution of polyvalentprotein mass spectrometry with MassLynx version 4.1 (from Waters).

[Table 1]

TABLE 1-1 Calculated Observed Ex- molecular molecular ample Compoundweight Observed ions (m/z) weight 1 S1C(disialo)- 5427.7 [M + 3H]³⁺1810.0 5427.0 SRIF28 [M + 4H]⁴⁺ 1357.5 [M + 5H]⁵⁺ 1086.4 2 N5C(disialo)-5400.6 [M + 3H]³⁺ 1801.0 5400.1 SRIF28 [M + 4H]⁴⁺ 1350.8 [M + 5H]⁵⁺1081.2 3 A9C(disialo)- 5443.7 [M + 3H]³⁺ 1815.4 5443.1 SRIF28 [M + 4H]⁴⁺1361.8 [M + 5H]⁵⁺ 1089.6 4 E12C(disialo)- 5385.6 [M + 3H]³⁺ 1796.05384.0 SRIF28 [M + 4H]⁴⁺ 1347.0 [M + 5H]⁵⁺ 1078.0 5 R13C(disialo)-5358.6 [M + 3H]³⁺ 1787.0 5358.0 SRIF28 [M + 4H]⁴⁺ 1340.5 [M + 5H]⁵⁺1072.6 6 K14C(disialo)- 5386.6 [M + 3H]³⁺ 1796.4 5386.0 SRIF28 [M +4H]⁴⁺ 1347.5 [M + 5H]⁵⁺ 1078.2 7 A15C(disialo)- 5443.7 [M + 3H]³⁺ 1815.45443.1 SRIF28 [M + 4H]⁴⁺ 1361.8 [M + 5H]⁵⁺ 1089.6 8 G16C(disialo)-5457.7 [M + 3H]³⁺ 1820.0 5457.1 SRIF28 [M + 4H]⁴⁺ 1365.3 [M + 5H]⁵⁺1092.2 9 K18C(disialo)- 5386.6 [M + 3H]³⁺ 1796.4 5386.1 SRIF28 [M +4H]⁴⁺ 1347.5 [M + 5H]⁵⁺ 1078.2 [M + 6H]⁶⁺ 898.7 [M + 7H]⁷⁺ 770.5

[Table 2]

TABLE 1-2 Calculated Observed Ex- molecular molecular ample Compoundweight Observed ions (m/z) weight 10 N19C(disialo)- 5400.6 [M + 3H]³⁺1801.0 5400.1 SRIF28 [M + 4H]⁴⁺ 1351.0 [M + 5H]⁵⁺ 1080.8 11F21C(disialo)- 5367.6 [M + 3H]³⁺ 1790.0 5366.9 SRIF28 [M + 4H]⁴⁺ 1342.7[M + 5H]⁵⁺ 1074.4 12 T26C(disialo)- 5413.6 [M + 3H]³⁺ 1354.3 5412.1SRIF28 [M + 4H]⁴⁺ 1083.4 [M + 5H]⁵⁺ 903.0 [M + 6H]⁶⁺ 774.3 1329C(disialo)- 5514.7 [M + 3H]³⁺ 1839.1 5514.1 SRIF28 [M + 4H]⁴⁺ 1379.5[M + 5H]⁵⁺ 1103.8 14 30C(disialo)- 5615.8 [M + 3H]³⁺ 1872.7 5615.1SRIF28 [M + 4H]⁴⁺ 1404.8 [M + 5H]⁵⁺ 1124.0 15 S1C(disialo)- 5427.7 [M +3H]³⁺ 1810.1 5427.2 D-Trp22- [M + 4H]⁴⁺ 1357.8 SRIF28 [M + 5H]⁵⁺ 1086.4[M + 6H]⁶⁺ 905.5 16 A9C(disialo)- 5443.7 [M + 3H]³⁺ 1815.4 5442.9D-Trp22- [M + 4H]⁴⁺ 1361.8 SRIF28 [M + 5H]⁵⁺ 1089.6 17 C(disialo)-4004.1 [M + 2H]²⁺ 2002.9 4003.3 SRIF14 [M + 3H]³⁺ 1335.4 [M + 4H]⁴⁺1001.8 18 C(disialo)- 4288.4 [M + 3H]³⁺ 1430.2 4287.5 R-K-SRIF14 [M +4H]⁴⁺ 1072.9 19 C(disialo)- 4201.4 [M + 3H]³⁺ 1401.2 4200.5 C12linker-[M + 4H]⁴⁺ 1051.2 SRIF14 [M + 5H]⁵⁺ 841.1

[Table 3]

TABLE 1-3 Calculated Observed Ex- molecular molecular ample Compoundweight Observed ions (m/z) weight 20 S1-2C(disialo)- 7793.8 [M + 4H]⁴⁺1949.4 7793.4 SRIF28 [M + 5H]⁵⁺ 1559.7 [M + 6H]⁶⁺ 1299.9 21 S1C(disialo)

7679.7 [M + 3H]³⁺ 1920.4 7678.7 N5C(disialo)- [M + 4H]⁴⁺ 1536.8 SRIF28[M + 5H]⁵⁺ 1280.9 22 S1C(disialo)

7637.7 [M + 3H]³⁺ 1910.2 7636.6 R13C(disialo)- [M + 4H]⁴⁺ 1528.3 SRIF28[M + 5H]⁵⁺ 1273.8 23 N5C(disialo)

7695.7 [M + 5H]⁵⁺ 1540.1 7695.2 A9C(disialo)- [M + 6H]⁶⁺ 1283.6 SRIF28[M + 7H]⁷⁺ 1100.3 [M + 8H]⁸⁺ 962.9 24 S1-3C(disialo)- 10160.0 [M + 3H]³⁺3387.6 10158.2 SRIF28 [M + 4H]⁴⁺ 2540.9 [M + 5H]⁵⁺ 2032.7 [M + 6H]⁶⁺1694.2 [M + 7H]⁷⁺ 1452.3 25 S1C(disialo)

9974.8 [M + 5H]⁵⁺ 1995.9 9974.3 N5C(disialo)

[M + 6H]⁶⁺ 1663.2 A9C(disialo)- SRIF28 26 S1C(mono- 5136.4 [M + 3H]³⁺1713.0 5134.9 sialo)- [M + 4H]⁴⁺ 1284.7 SRIF28 [M + 5H]⁵⁺ 1028.2 [M +6H]⁶⁺ 856.8

indicates data missing or illegible when filed

[Table 4]

TABLE 1-4 Calculated Observed Ex- molecular molecular ample Compoundweight Observed ions (m/z) weight 27 S1C(asialo)- 4845.1 [M + 3H]³⁺2615.7 4843.9 SRIF28 [M + 4H]⁴⁺ 1212.0 [M + 5H]⁵⁺ 969.8 28S1-2C(asialo)- 6628.8 [M + 4H]⁴⁺ 1658.1 6627.2 SRIF28 [M + 5H]⁵⁺ 1326.7[M + 6H]⁶⁺ 1105.6 [M + 7H]⁷⁺ 947.8 [M + 8H]⁸⁺ 829.4 29 S1-3C(asialo)-8412.5 [M + 4H]⁴⁺ 2103.7 8411.7 SRIF28 [M + 5H]⁵⁺ 1683.3 [M + 6H]⁶⁺1403.0 30 N5N(disialo)- 5354.5 [M + 3H]³⁺ 1785.7 5354.0 SRIF28 [M +4H]⁴⁺ 1339.5 [M + 5H]⁵⁺ 1071.8 31 S1N(disialo)- 5381.6 [M + 3H]³⁺ 1794.75380.9 SRIF28 [M + 4H]⁴⁺ 1346.2 [M + 5H]⁵⁺ 1077.2 32 S1C(disialo)

5676.9 [M + 3H]³⁺ 1893.5 5676.4 N19C(GlcNAc)- [M + 4H]⁴⁺ 1420.1 SRIF28[M + 5H]⁵⁺ 1136.3 33 S1C(disialo)

6366.6 [M + 3H]³⁺ 2122.9 6366.7 N19C(diMan)

[M + 4H]⁴⁺ 1592.5 SRIF28 [M + 5H]⁵⁺ 1274.4

indicates data missing or illegible when filed

[Table 5]

TABLE 1-5 Calculated Observed Ex- molecular molecular ample Compoundweight Observed ions (m/z) weight 34 C(disialo)- 5514.7 [M + 4H]⁴⁺1379.7 5514.6 SRIF28 [M + 5H]⁵⁺ 1103.9 [M + 6H]⁶⁺ 920.1 [M + 7H]⁷⁺ 788.835 R11C(disialo)- 5358.5 [M + 3H]³⁺ 1787.4 5358.6 SRIF28 [M + 4H]⁴⁺1340.7 [M + 5H]⁵⁺ 1072.7 36 F20C(disialo)- 5367.6 [M + 3H]³⁺ 1790.15367.1 SRIF28 [M + 4H]⁴⁺ 1342.8 [M + 5H]⁵⁺ 1074.4 37 T24C(disialo)-5413.6 [M + 3H]³⁺ 1805.7 5412.6 SRIF28 [M + 4H]⁴⁺ 1354.2 [M + 5H]⁵⁺1083.7 38 F25C(disialo)- 5367.6 [M + 3H]³⁺ 1790.1 5367.1 SRIF28 [M +4H]⁴⁺ 1342.8 [M + 5H]⁵⁺ 1074.4 [M + 6H]⁶⁺ 895.4 39 S27C(disialo)- 5427.7[M + 3H]³⁺ 1810.1 5427.4 SRIF28 [M + 4H]⁴⁺ 1357.8 40 C(disialo)- 4131.2[M + 2H]²⁺ 2066.9 4131.7 K-SRIF14 [M + 3H]³⁺ 1378.2 [M + 4H]⁴⁺ 1033.9 41S1C(disialo)- 5443.7 [M + 3H]³⁺ 1815.4 5443.1 F25Y-SRIF28 [M + 4H]⁴⁺1361.6 [M + 5H]⁵⁺ 1089.6 42 S1C(disialo)- 5426.7 [M + 3H]³⁺ 1809.85426.2 SRIF28-amide [M + 4H]⁴⁺ 1357.6 [M + 5H]⁵⁺ 1086.2 [M + 6H]⁶⁺ 905.243 C(disialo)- 4207.3 [M + 3H]³⁺ 1403.2 4206.6 PEGlinker- [M + 4H]⁴⁺1052.7 SRIF14 [M + 5H]⁵⁺ 842.3 44 Biotin- 5654.0 [M + 3H]³⁺ 1885.45653.2 S1C(disialo)- [M + 4H]⁴⁺ 1414.3 SRIF28 [M + 5H]⁵⁺ 1131.7 [M +6H]⁶⁺ 943.2 45 Biotin- 5857.2 [M + 3H]³⁺ 1953.2 5856.4 PEGlinker- [M +4H]⁴⁺ 1465.1 S1C(disialo)- [M + 5H]⁵⁺ 1172.1 SRIF28 [M + 6H]⁶⁺ 977.1[M + 7H]⁷⁺ 837.6

[Table 6]

TABLE 1-6 Calculated Observed Ex- molecular molecular ample Compoundweight Observed ions (m/z) weight 46 Azido-S1C(disialo)-SRIF28 5552.8[M + 3H]³⁺ 1851.8 5552.3 [M + 4H]⁴⁺ 1389.1 [M + 5H]⁵⁺ 1111.7 47S1C(disialo)

E12C(disialo)- 7664.7 [M + 4H]⁴⁺ 1917.1 7664.1 SRIF28 [M + 5H]⁵⁺ 1533.8[M + 6H]⁶⁺ 1278.4 48 2C(disialo)-R-K-SRIF14 6654.6 [M + 3H]³⁺ 2219.26653.4 [M + 4H]⁴⁺ 1664.4 [M + 5H]⁵⁺ 1331.9 [M + 6H]⁶⁺ 1109.9 493C(disialo)-R-K-SRIF14 9020.8 [M + 4H]⁴⁺ 2256.1 9020.2 [M + 5H]⁵⁺ 1805.1[M + 6H]⁶⁺ 1504.4 50 S1C(diGlcNAc)-SRIF28 4520.9 [M + 3H]³⁺ 1507.74519.9 [M + 4H]⁴⁺ 1131.0 [M + 5H]⁵⁺ 905.0 51 S1C(diMan)-SRIF28 4114.5[M + 3H]³⁺ 1372.3 4113.8 [M + 4H]⁴⁺ 1029.5 [M + 5H]⁵⁺ 823.8 52N19C(diMan)-SRIF28 4087.5 [M + 3H]³⁺ 1363.3 4086.8 [M + 4H]⁴⁺ 1023.0 53S1C(GlcNAc)-SRIF28 3424.9 [M + 2H]²⁺ 1713.3 3424.5 [M + 3H]³⁺ 1142.5[M + 4H]⁴⁺ 856.9 [M + 5H]⁵⁺ 686.1 54 N19C(GlcNAc)-SRIF28 3397.8 [M +2H]²⁺ 1699.8 3397.5 [M + 3H]³⁺ 1133.5 [M + 4H]⁴⁺ 850.4 55S1C(trisialo)-SRIF28 6084.2 [M + 3H]³⁺ 2028.8 6083.4 [M + 4H]⁴⁺ 1521.9[M + 5H]⁵⁺ 1217.7 [M + 6H]⁶⁺ 1014.9 56 S1C(tetrasialo)-SRIF28 6740.8[M + 3H]³⁺ 2247.7 6739.6 [M + 4H]⁴⁺ 1685.9 [M + 5H]⁵⁺ 1349.1 [M + 6H]⁶⁺1124.3 [M + 7H]⁷⁺ 963.8 57 S1C(disialo(aminoethylamide))- 5511.8 [M +3H]³⁺ 1838.0 5511.1 SRIF28 [M + 4H]⁴⁺ 1378.5 [M + 5H]⁵⁺ 1103.2 [M +6H]⁶⁺ 919.5 [M + 7H]⁷⁺ 788.2

indicates data missing or illegible when filed

[Table 7]

TABLE 1-7 Calculated Observed Ex- molecular molecular ample Compoundweight Observed ions (m/z) weight 58 S1C(disialo(amide))-SRIF28 5425.7[M + 3H]³⁺ 1809.3 5423.9 [M + 4H]⁴⁺ 1357.0 [M + 5H]⁵⁺ 1085.8 [M + 6H]⁶⁺905.2 59 S1C(disialo(Bn))-SRIF28 5607.9 [M + 3H]³⁺ 1870.0 5605.9 [M +4H]⁴⁺ 1402.5 [M + 5H]⁵⁺ 1122.2 [M + 6H]⁶⁺ 935.5 60S1C(disialo(hexadecylamide))-SRIF28 5874.5 [M + 3H]³⁺ 1958.9 5873.6 [M +4H]⁴⁺ 1469.4 [M + 5H]⁵⁺ 1175.7 [M + 6H]⁶⁺ 979.9 61S1-2C(disialo(amide))-SRIF28 7789.9 [M + 4H]⁴⁺ 1948.2 7789.9 [M + 5H]⁵⁺1559.0 [M + 6H]⁶⁺ 1299.2 62 S1-2C(disialo(Bn))-SRIF28 8154.3 [M + 4H]⁴⁺2039.3 8153.0 [M + 5H]⁵⁺ 1631.6 [M + 6H]⁶⁺ 1360.2 63S1C(Asn(disialo))-SRIF28 5542.7 [M + 3H]³⁺ 1848.4 5542.2 [M + 4H]⁴⁺1386.6 [M + 5H]⁵⁺ 1109.4 [M + 6H]⁶⁺ 924.9 64 S1N(disialo)

N19C(diMan)-SRIF28 6320.5 [M + 3H]³⁺ 2107.9 6319.5 [M + 4H]⁴⁺ 1580.9[M + 5H]⁵⁺ 1265.1 65 C(disialo(aminoethylamide))

7878.0 [M + 4H]⁴⁺ 1970.3 7878.1 S1C(disialo)-SRIF28 [M + 5H]⁵⁺ 1576.4[M + 6H]⁶⁺ 1313.9 [M + 7H]⁷⁺ 1126.4 [M + 8H]⁸⁺ 985.5 [M + 9H]⁹⁺ 876.1 66S1-4C(disialo)-SRIF28 12526.2 [M + 5H]⁵⁺ 2506.1 12525.5 [M + 6H]⁶⁺2088.6 [M + 7H]⁷⁺ 1790.4

indicates data missing or illegible when filed

Example 67-1 Calculation of Receptor Binding Affinity

Competitive binding assay was performed with the method below tocalculate receptor binding affinity.

Reagents employed in the competitive binding assay and their properchemical names are as follows: HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and BSA (bovineserum albumin).

Competitive binding assay was consigned to Ricerca Biosciences, andexperiment and data analysis was performed. The receptor subtypes andreceptor membrane samples employed are shown in Table 2. Common in eachbinding assay, [¹²⁵I]Tyr¹¹-Somatostatin 14 (Tyr11-SRIF14) was used asthe labeled ligand, Somatostatin-14 (SRIF14) was used as the unlabeledligand, and 25 mM HEPES containing 5 mM MgCl₂, 1 mM CaCl₂, and 0.1% BSA,pH 7.4 was used as the buffer. The test substance was used at aconcentration of 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM, andmixed with the membrane sample into a reaction solution. Moreover, theconcentrations of the labeled and unlabeled ligands added to thereaction solution are shown in Table 2. The incubation condition of thereaction solution was at 25° C. for 4 hours for SSTR2 and at 25° C. for2 hours for SSTR1, SSTR3, SSTR4, and SSTR5. For each experiment run,SRIF14 was used as the positive control. For data analysis, 50%inhibitory concentration (IC₅₀ value) was determined using MathIQ™ (IDBusiness Solutions, UK) in a nonlinear least squares method based on thenumerical data of the binding inhibition rate. The binding inhibitionconstant (Ki value) was calculated by the method of Cheng, Y et al.(Biochem Pharmacol, 22, 3099-3108, 1973).

[Table 8]

TABLE 2 SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 Labeled 0.1 nM 0.03 nM 0.1 nM 0.1nM 0.1 nM ligand Unlabeled 1 μM 1 μM 1 μM 1 μM 1 μM ligand Source HumanHuman Human Human Human recombinant recombinant recombinant recombinantrecombinant CHO-K1 cells CHO-K1 cells CHO-K1 cells Chem-1 cells Chem-1cells

Compounds subjected to binding assay and the results of the bindingexperiment are shown in Table 3A. Moreover, octreotide, SRIF14 andSRIF28 were similarly evaluated as control compounds. Note that foroctreotide, IC₅₀ values could not be calculated for SSTR1 and SSTR4since the maximum concentration was 100 nM, and is thus shown as >100nM.

FIGS. 1A and 1B show examples of the structures of glycosylated peptides(glycosylated forms) corresponding to the compound names in Table 3A.

[Table 9]

TABLE 3A SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 Example IC₅₀ (nM) Ki (nM) IC₅₀(nM) Ki (nM) IC₅₀ (nM) Ki (nM) IC₅₀ (nM) Ki (nM) IC₅₀ (nM) Ki (nM)  1S1C(disialo)-SRIF28 2.19 1.81 0.109 0.0576 0.480 0.164 2.30 2.07 0.4420.250  2 N5C(disialo)-SRIF28 1.10 0.912 0.0945 0.0502 0.278 0.0950 1.301.17 0.458 0.259  3 A9C(disialo)-SRIF28 2.08 1.72 0.0741 0.0394 0.5930.203 1.72 1.54 0.471 0.266  4 E12C(disialo)-SRIF28 1.70 1.40 0.1120.0598 0.362 0.124 2.04 1.84 0.376 0.212  5 R13C(disialo)-SRIF28 15.413.6 0.310 0.165 2.87 0.981 6.43 5.79 3.28 1.85  6 K14C(disialo)-SRIF2813.8 11.4 0.481 0.256 2.59 0.887 8.02 7.22 4.01 2.27 10N19C(disialo)-SRIF28 15.1 13.3 1.21 0.615 1.17 0.603 12.2 11.0 3.69 2.0913 29C(disialo)-SRIF28 4.43 3.67 0.738 0.392 7.00 2.39 6.11 5.50 3.732.11 14 30C(disialo)-SRIF28 4.21 3.48 0.912 0.275 6.91 2.36 2.53 2.281.86 1.05 15 S1C(disialo)-D-Trp22-SRIF28 2.23 1.85 0.0324 0.0172 0.8340.285 5.85 5.26 0.565 0.319 16 A9C(disialo)-D-Trp22- 6.92 6.73 0.03960.0210 0.256 0.0876 4.87 4.38 0.194 0.110 SRIF28 17 C(disialo)-SRIF1432.3 26.7 6.336 0.179 2.62 1.58 8.24 7.41 5.70 3.22 18C(disialo)-R-K-SRIF14 3.09 2.56 0.104 0.0551 0.231 0.0791 1.06 0.9510.187 0.106 19 C(disialo)-C12linker-SRIF14 15.5 12.8 0.502 0.267 3.121.07 2.92 2.62 2.29 1.29 20 S1-2C(disialo)-SRIF28 18.8 15.5 0.338 0.1791.28 0.439 5.61 5.05 0.607 0.343 21 S1C(disialo)•N5C(disialo)- 6.89 5.700.224 0.119 1.58 0.539 4.00 3.60 0.728 0.411 SRIF28 22S1C(disialo)•R13C(disialo)- 177 147 6.28 0.33 11.0 3.77 45.0 40.5 14.78.33 SRIF28 23 N5C(disialo)•A9C(disialo)- 30.0 24.9 0.793 0.421 4.091.49 14.2 12.8 1.81 1.02 SRIF28 24 S1-3C(disialo)-SRIF28 20.4 16.9 0.4210.224 3.30 1.13 7.69 6.92 0.927 0.524 25 S1C(disialo)•N5C(disialo)- 69.257.3 0.985 0.523 7.93 2.71 25.2 22.7 4.14 2.34 A9C(disialo)-SRIF28 26S1C(monosialo)-SRIF28 1.43 1.19 0.0465 0.0247 0.322 0.110 1.14 1.030.372 0.210 27 S1C(asialo)-SRIF28 0.851 0.704 0.0224 0.0119 0.155 0.05310.627 0.504 0.460 0.260 28 S1-2C(asialo)-SRIF28 0.860 0.712 0.06320.0336 0.177 0.0604 0.780 0.702 0.190 0.107 29 S1-3C(asialo)-SRIF28 4.213.49 0.0978 0.0519 0.581 0.199 1.44 1.29 0.369 0.209 30N5N(disialo)-SRIF28 3.02 2.50 0.0685 0.0364 0.579 0.198 2.86 2.57 0.4650.263 31 S1N(disialo)-SRIF28 2.68 2.21 0.0764 0.0401 0.314 0.107 1.070.260 0.272 0.154 — SRIF28 0.433 0.962 0.0142 0.00755 0.131 0.0450 0.3000.278 0.578 0.326 — Octreotide >100 — 0.0628 0.0334 6.41 2.19 >100 —9.39 5.31 — SRIF28 0.353 0.292 0.0271 0.0144 0.0368 0.0297 0.294 0.2050.423 0.239

The control octreotide bound to each of receptors SSTR2, SSTR3, andSSTR5, and SRIF14 and SRIF28 bound to all SSTRs. As shown in Table 3A,the compounds according to the present invention potently bound to allSSTRs. The binding affinity of the positive control SRIF14 against SSTR1in terms of Ki value was 0.362 nM, while compounds having one or twosugar chain(s) of the present invention was 0.704-147 nM, showingsufficient receptor binding affinity. Moreover, the compounds of thepresent invention having three sugar chains (the compounds of Examples24, 25, 29) also had sufficient receptor binding affinity, showing 3.49nM, 16.9 nM, and 57.3 nM. Since bioavailability (BA) will beconsiderably increased due to the extension of its half-life in blood,they can work effectively on receptors in vivo even if the Ki value ofthe binding affinity is somewhat high. Similarly, where the bindingaffinity of SRIF14 against SSTR2, SSTR3, and SSTR4 in terms of Ki valuewere each 0.00755 nM, 0.0450 nM, and 0.273 nM, the compounds of thepresent invention were each 0.0119-3.33 nM, 0.0531-3.77 nM, and0.564-40.5 nM, all showing sufficient receptor binding affinity.Moreover, where the binding affinity of SRIF14 against SSTR5 was 0.326nM, the compounds of the present invention showed a sufficient receptorbinding affinity of up to 8.33 nM.

The compounds of Examples 1-6, 10, 13-19, 26, 27, 30 and 31 were foundto be monoglycosylated modified forms having affinity towards allreceptors: SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5. Similarly, it was alsofound that Examples 20-23 and 28 are diglycosylated modified formshaving affinity towards all receptors SSTR1-SSTR5, and Examples 24, 25,and 29 are triglycosylated modified forms having affinity towards allreceptors SSTR1-SSTR5.

Example 67-2 Calculation of Receptor Binding Affinity—2

Competitive binding assay was performed with each compound shown inTable 3B with the method described in Example 67-1, and receptor bindingaffinity was calculated. Moreover, SRIF14 and SRIF28 were similarlyevaluated as control compounds. The results of the binding assay areshown in Table 3B.

FIGS. 1C and 1D show examples of the structures of glycosylated peptidescorresponding to the compound names in Table 3B.

[Table 10]

TABLE 3B Exam- SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 ple IC₅₀ (nM) Ki (nM) IC₅₀(nM) Ki (nM) IC₅₀ (nM) Ki (nM) IC₅₀ (nM) Ki (nM) IC₅₀ (nM) Ki (nM)  1S1C(disialo)-SRIF28 7.71 6.45 0.543 0.289 1.08 0.676 7.10 6.39 1.610.011  7 A15C(disialo)-SRIF28 11.4 9.43 0.938 0.498 3.49 1.19 8.18 7.303.84 2.17  8 G16C(disialo)-SRIF28 17.4 14.4 1.22 0.646 3.12 1.07 12.211.0 5.67 3.21  9 K18C(disialo)-SRIF28 45.4 37.6 12.2 6.51 14.7 5.0362.7 56.5 160 95.0 12 T26C(disialo)-SRIF28 21.6 17.9 310 160 21.7 7.4426.8 24.1 480 230 26 S1C(monosialo)-SRIF28 5.38 4.46 0.213 0.112 1.180.405 2.35 2.12 0.816 0.460 27 S1C(asialo)-SRIF28 3.97 3.29 0.200 0.1051.02 0.349 2.98 2.68 1.04 0.588 32 S1C(disialo)•N19C(GlcNAc)- <10 <8.272.20 1.17 3.05 1.04 8.55 7.69 2.37 1.34 SRIF28 33S1C(disialo)•N19C(diMan)- 440 37.0 3.73 1.98 3.63 1.24 25.0 23.0 5.693.22 SRIF28 34 C(disialo)-SRIF28 5.57 4.61 0.478 0.255 2.55 0.870 8.657.79 2.62 1.48 35 R11C(disialo)-SRIF28 22.0 18.2 1.38 0.735 6.34 2.1720.2 18.1 3.65 2.07 38F25C(disialo)-SRIF28 >1000 >828 >1000 >531 >1000 >342 130 110 53.0 33.039 S27C(disialo)-SRIF28 28.4 23.5 0.326 0.170 6.77 2.31 33.4 30.1 4.942.80 40 C(disialo)-K-SRIF14 20.7 17.2 1.97 1.05 3.55 1.21 11.0 9.77 3.521.99 41 S1C(disialo)-F25Y-SRIF28 1.77 1.46 0.310 0.160 0.480 0.160 1.551.39 1.09 0.620 42 S1C(disialo)-SRIF28-amide 4.87 4.03 1.32 0.700 5.912.02 6.88 6.19 3.75 2.12 43 C(disialo)-PEGlinker-SRIF14 36.0 30.0 4.672.48 8.71 2.98 21.0 19.0 >10 >5.65 44 Biotin-S1C(disialo)-SRIF28 11.09.05 1.68 0.890 2.92 1.00 9.28 8.35 2.42 1.37 45 Biotin-PEGlinker- 9.417.39 1.86 1.04 3.41 1.17 9.31 8.38 2.00 1.13 S1C(disialo)-SRIF28 46azido-S1C(disialo)-SRIF28 8.82 7.30 1.29 0.690 3.17 1.09 5.34 4.81 0.8400.470 47 S1C(disialo)•E12C(disialo)- 22.0 19.0 2.70 1.44 4.62 1.58 20.018.0 2.11 1.19 SRIF28 48 2C(disialo)-RK-SRIF14 48.0 48.0 5.35 2.84 9.743.33 49.0 44.0 6.16 3.48 49 3C(disialo)-RK-SRIF14 270 230 32.0 17.0 40.014.0 >100 >90.0 41.0 23.0 50 S1C(diGlcNAc)-SRIF28 5.04 4.17 0.190 0.1002.02 0.690 3.91 3.52 1.45 0.820 51 S1C(dMan)-SRIF28 5.06 4.18 0.1400.0750 1.32 0.450 3.01 2.70 0.550 0.310 52 N19C(diMan)-SRIF28 6.58 5.441.17 0.620 1.55 0.530 6.02 7.22 2.94 1.66 53 S1C(GlcNAc)-SRIF28 5.914.89 0.225 0.120 1.65 0.555 2.89 2.60 1.15 0.650 54 N19C(GlcNAc)-SRIF282.60 2.15 0.940 0.500 <1 <0.342 2.38 2.15 1.89 1.07 55S1C(trisialo)-SRIF28 17.0 14.0 0.780 0.410 5.59 1.91 15.0 14.0 1.95 1.1056 S1C(tetrasialo)-SRIF28 17.3 14.2 1.12 0.597 5.63 1.93 14.3 12.6 2.121.20 57 S1C(disialo(amino- 2.35 1.94 0.101 0.0536 0.766 0.264 2.24 2.012.08 1.18 ethylamide))-SRIF28 58 S1C(disialo(amide))-SRIF28 5.09 4.210.207 0.110 1.53 0.522 6.12 5.50 2.60 1.47 59 S1C(disialo(Bn))-SRIF286.20 5.12 0.247 0.130 2.15 0.733 6.01 5.41 3.25 1.83 60S1C(disialo(hexadexyl- 5.53 4.57 1.04 0.550 11.0 3.75 26.0 24.0 37.021.0 amide))-SRIF28 61 S1-2C(disialo(amide))- 6.92 5.73 0.830 0.440 2.060.710 4.76 4.28 2.67 1.51 SRIF28 62 S1-2C(disialo(Bn))-SRIF28 6.01 4.970.830 0.440 2.58 0.880 8.20 7.38 2.60 1.47 63 S1C(Asn(disialo))-SRIF2815.0 13.0 0.610 0.330 5.57 1.91 16.0 14.0 1.61 0.910 64S1N(disialo)•N19C(diMan)- 35.0 29.0 5.65 3.00 3.90 1.33 25.0 22.0 8.534.82 SRIF28 65 C(disialo(amino- 6.20 5.13 0.960 0.510 1.89 0.650 6.125.51 1.81 1.02 ethylamide))•S1C(disialo)- SRIF28 66S1-4C(disialo)-SRIF28 130 100 3.76 2.00 10.0 3.43 66.0 59.0 10.0 5.82 —SRIF28 0.533 1.80 0.680 0.565 0.206 0.116 0.776 1.23 0.189 0.666 —SRIF14 1.25 2.69 0.0329 0.0993 0.0697 0.403 0.823 1.95 0.419 0.813

As shown in Table 3B, the controls SRIF14 and SRIF28 bound to allreceptors SSTR1-SSTR5. Since the number of test runs etc. was differentfrom Example 67-1, the Ki value of SRIF14 against each receptor wasdifferent at a high value of 2.5 to 13.2. The Ki values of the compoundsof Examples 1, 7, 8, 9, 26, 27, 32, 33, 34, 35, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, and 66 against SSTR1 were 1.46-100 nM, and similarly 0.0536-6.51nM for SSTR2, 0.160-7.44 nM for SSTR3, 1.39-59.0 nM for SSTR4, and0.310-95.0 nM for SSTR5, showing high binding affinity towards all thereceptors. The Ki values of the compound of Example 12 against SSTR2 andSSTR5 were 280-1600-folds that of SRIF14 but the Ki values againstSSTR1, SSTR3, and SSTR4 were 17.9, 7.44, and 24.1 nM, and it was thoughtthat binding affinity was retained. The affinity of the compound ofExample 38 against SSTR1, SSTR2, and SSTR3 was significantly reducedwith Ki values of 310-5300-folds that of SRIF14 but the Ki valuesagainst SSTR4 and SSTR5 were 110 and 33.0 nM, and it was thought thatbinding affinity was retained. The Ki values of the compound of Example49 against SSTR2 and SSTR4 were 170-folds and 46-folds or more that ofSRIF14 but the Ki values against SSTR1, SSTR3, and SSTR5 were each 270nM, 14.0 nM, and 23.0 nM, and it was thought that binding affinity wasretained.

Example 67-3 Agonist Activity Evaluation Employing Receptor ExpressionCells—1

Somatostatin receptor is a G-protein-coupled receptor (GPCR).SSTR1-SSTR5 all suppress adenyl cyclase activity via G-protein subfamilyGi/Go and reduces intracellular cAMP concentration. In this experimentalline, each somatostatin receptor expression cells was employed tocalculate the IC₅₀ value of the cAMP production suppressing action ofthe test substance to evaluate agonist activity. Moreover, SRIF14,SRIF28 were similarly evaluated as control compounds.

Reagents employed in the present experiment and their proper chemicalnames are as follows: DMEM (Dulbecco's Modified Eagle's Medium), IBMX(3-isobutyl-1-methylxanthine), and HBSS (Hank's Buffered Salt Solution).

For evaluations of SSTR2-SSTR5, experiments were performed under thefollowing conditions. DMEM containing 0.3% BSA and 0.5 mM IBMX wasemployed as the buffer, and receptor expression cells shown in Table 3Cwere seeded at 10⁴ cells/well. The test substance was treated by mixingat a concentration of 0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM,1 nM, 10 nM, 100 nM, or 1000 nM with 10 μM forskolin, and allowed toreact at room temperature for 30 minutes. After the reaction, the cellswere dissolved with 0.2 N HCl, and the amount of cAMP accumulated in thecell was measured with Cayman cyclic AMP EIA kit (Cayman, 582002).

Evaluation of SSTR1 was consigned to Cerep, and the experiment wasperformed. HBSS containing 20 mM HEPES (pH 7.4) and 500 μM IBMX was usedas the buffer, and SSTR1 receptor expression cells were seeded at 10⁴cells/well. The test substance was treated by mixing at a concentrationof 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM with 1 μM NKH477,and allowed to react at 37° C. for 20 minutes. After the reaction, theamount of cAMP accumulated in the cell was measured with Cisbio cAMPdynamic2 kit (Cisbio, 62AM4PE).

[Table 11]

TABLE 3C SSTR2 SSTR3 SSTR4 SSTR5 Distributer Perkin Elmer Serial NumberES-521-CF ES-523-CF ES-524-CF ES-522-CF Derived cell CHO-K1 cells

The experimental results (IC₅₀ (nM)) of the agonist activity evaluationtest when employing the compounds of Examples 1, 18, 27, 28, 57, and 58as the glycosylated form are shown in Table 3D and FIG. 1E. IC₅₀ of thecontrol compounds SRIF14 and SRIF28 was 0.83-0.89 nM against SSTR1, and0.0076-0.073 nM, 0.029-0.21 nM, 0.015-0.074 nM, and 0.066-0.12 nMagainst SSTR2, SSTR3, SSTR4, and SSTR5, respectively. IC₅₀ of theagonist activity of the glycosylated compounds against SSTR1, SSTR2,SSTR3, SSTR4, and SSTR5 were 1.0-2.4 nM, 0.041-0.10 nM, 0.12-0.30 nM,0.039-0.085 nM, and 0.043-0.081 nM, respectively, and potent agonistactivity was shown against all receptors SSTR1-SSTR5. From the resultsshown in Examples 67-1 and 67-2, it is apparent that these compoundshave receptor binding affinity, and it became clear that these compoundsexert proper agonistic action by binding to a receptor.

TABLE 3D Example SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 — SRIF14 0.83 ± 0.520.0076 ± 0.0012 0.029 ± 0.017 0.015 ± 0.006 0.12 ± 0.03 — SRIF28 0.89 ±0.41 0.073 ± 0.014 0.21 ± 0.08 0.074 ± 0.058 0.066 ± 0.033  1S1C(disialo)-SRIF28 2.2 ± 1.1 0.075 ± 0.030 0.13 ± 0.06 0.065 ± 0.0420.056 ± 0.028 18 Cys(disialo)-R-K-SRIF14 1.2 ± 0.6 0.055 ± 0.015 0.18 ±0.10 0.050 ± 0.025 0.052 ± 0.032 27 S1C(asialo)-SRIF28 1.6 ± 0.6 0.041 ±0.006 0.12 ± 0.05 0.039 ± 0.021 0.055 ± 0.031 28 S1-2C(asialo)-SRIF281.5 ± 0.6 0.079 ± 0.033 0.15 ± 0.08 0.051 ± 0.046 0.064 ± 0.029 57S1C(disialo(aminoethylamide))-SRIF28 1.0 ± 0.9 0.10 ± 0.02 0.30 ± 0.150.085 ± 0.011 0.081 ± 0.019 58 S1C(disialo(amide))-SRIF28 2.4 ± 1.70.055 ± 0.013 0.14 ± 0.06 0.043 ± 0.023 0.043 ± 0.015

Example 67-4 Agonist Activity Evaluation Employing Receptor ExpressionCells—2

Agonist activity evaluation test was carried out similarly to Example67-3 employing each of the compounds described in Table 3E as theglycosylated form. The experimental results are shown in Table 3E. InTable 3E, fields with “- (hyphen)” shown indicates that the test was notperformed.

[Table 13]

TABLE 3E Experiment SSTR1 SSTR2 SSTR3 SSTR4 SSTR5 — SRIF14 0.83 0.00760.029 0.015 0.12 — SRIF28 0.89 0.073 0.21 0.074 0.066  4E12C(disialo)-SRIF28 2.3 — — — — 10 N19C(disialo)-SRIF28 22 — — — — 15S1C(disialo)-D-Trp22-SRIF28 3.5 — — — — 17 C(disialo)-SRIF14 — 0.0290.18 0.10 0.23 19 C(disialo)-C12linker-SRIF14 — — — 0.11 — 20S1-2C(disialo)-SRIF28 — — 0.55 0.14 0.22 21S1C(disialo)•N5C(disialo)-SRIF28 — 0.072 0.37 0.041 0.090 23N5C(disialo)•A9C(disialo)-SRIF28 — 0.096 0.29 0.091 0.13 24S1-3C(disialo)-SRIF28 7.5 0.19 0.79 0.36 0.97 25S1C(disialo)•N5C(disialo)•A9C(disialo)-SRIF28 — 1.4 2.4 0.16 1.1 26S1C(monosialo)-SRIF28 1.5 0.059 0.15 0.046 0.062 29 S1-3C(asialo)-SRIF28— 0.19 — 0.10 — 31 S1N(disialo)-SRIF28 2.3 — — — — 32S1C(disialo)•N19C(GlcNac)-SRIF28 — 0.080 0.039 0.033 0.088 40C(disialo)-K-SRIF14 — 0.019 0.10 0.059 — 42 S1C(disialo)-SRIF28-amide1.7 — — — — 45 C(disialo)-PEG2linker-SRIF14 — 0.034 0.15 0.066 0.16 482C(disialo)-R-K-SRIF14 — 0.15 0.55 1.1 — 49 3C(disialo)-R-K-SRIF14 — 1.43.8 1.6 — 50 S1C(diGlcNAc)-SRIF28 1.2 0.045 0.14 0.050 0.033 51S1C(diMan)-SRIF28 1.2 0.035 0.11 0.043 0.037 52 N19C(diMan)-SRIF28 — — —0.11 — 53 S1C(GlcNAc)-SRIF28 1.4 0.040 0.14 0.049 0.031 54N19C(GlcNAc)-SRIF28 — — — 0.092 — 55 S1C(trisialo)-SRIF28 4.9 0.11 0.380.092 0.090 56 S1C(tetrasialo)-SRIF28 2.8 0.13 0.25 0.081 0.11 59S1C(disialo(Bn))-SRIF28 1.4 — — — — 60S1C(disialo(hexadecylamide))-SRIF28 1.3 — — — — 63S1C(Asn(disialo))-SRIF28 2.6 — — 0.13 — 65C(disialo(aminoethylamide))•S1C(disialo)-SRIF28 — 0.039 0.17 0.047 0.034(—: Not performed)

In Table 3E, IC₅₀ of the agonist activity of the glycosylated compoundsagainst SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 were 1.2-22 nM, 0.019-1.4nM, 0.039-3.8 nM, 0.033-1.6 nM, and 0.031-1.1 nM, respectively, andagonist activity was shown against receptors SSTR1-SSTR5. From theresults shown in Examples 67-1 and 67-2, it became clear that thesecompounds have receptor binding affinity, and these compounds exertagonist activity by binding to a receptor.

Example 68 Pharmacokinetics Test with Rats 1

In order to confirm that the glycosylated polypeptide of the presentinvention (glycosylated form) had improved pharmacokinetics profilessuch as drug plasma concentration—area under the time curve (AUC),half-life in blood (t_(1/2)), mean retention time (MRT), andbioavailability compared to a non-glycosylated SRIF28, pharmacokineticsanalysis by intravenous and subcutaneous administrations was performedwith rats.

68-1 Preparation of Administration Solution and Reagent

The glycosylated form (S1C(disialo)-SRIF28) was dissolved in JapanesePharmacopeia saline (from Otsuka Pharmaceutical factory, Inc.) toprepare a 40 μM solution to make an administration solution. PBSsolution was prepared by dissolving 1 tablet of Phosphate bufferedsaline (P4417 from Sigma) in 200 mL of ultrapure water. EDTA-PBS wasprepared by dissolving EDTA-2Na (from Wako Pure Chemical Industries,Ltd.) in PBS to 2 mg/mL. Aprotinin-containing EDTA-PBS solution wasprepared by dissolving aprotinin (010-11834 from Wako Pure ChemicalIndustries, Ltd.) in EDTA-PBS to 0.142 mg/mL, and was employed as ananticoagulant for collected blood.

68-2 Preparation of Plasma Sample

To the tail vain or dorsal subcutaneous of male SD rats (Crl: CD (SD),Charles River Japan, 6 weeks-old, n=3, body weight 161.3-239.3 g), theadministration solution prepared in the above 68-1 was administeredunder nonfasting condition at a dosage of 1 mL/kg with a glass syringeand a 26 G injection needle (all from Terumo Corporation) (40 nmol/kg asS1C(disialo)-SRIF28). Blood was collected from the rat cervical veinbefore administration, as well as at 2 minutes, 5 minutes, 15 minutes,30 minutes, 1 hour, 2 hours, 4 hours, and 8 hours after administration.0.2 mL of the collected blood was promptly mixed with 0.2 mL of theaprotinin-containing EDTA-PBS solution prepared in the above 68-1, andleft on ice for 30 minutes or more. After centrifugal separation(1,870×g, 4° C., 10 minutes), 250 μL of the supernatant was taken as theplasma sample. As blank plasma, plasma obtained by similarly treatingthe blood collected from untreated rat cervical vein was employed.Plasma samples were frozen in storage until employed for measurement.Tips and tubes used were low absorbent products from BM Equipment Co.,Ltd.

68-3 Measurement of Concentration in Plasma

Measurement of plasma concentration of S1C(disialo)-SRIF28 in the plasmasample obtained in the above 68-2 was performed with PhoenixPharmaceuticals somatostatin EIA kit (Phoenix Pharmaceuticals Inc,EK-060-03). The plasma sample was diluted with the assay buffer suppliedin the EIA kit to 5, 20, 100, 400, and 1600-folds as measurementsamples. The standard solution for creating a standard curve wasprepared as follows. First, the blank plasma obtained in the above 68-2was diluted with the assay buffer supplied in the EIA kit similarly tothe plasma sample, and this was used as the assay buffer for standardsolution preparation (for example, when diluting the plasma sample to100-fold, 1/100 amount of blank plasma was added to the assay buffersupplied in the EIA kit, and this was used as the assay buffer forstandard solution preparation). S1C(disialo)-SRIF28 was diluted with PBSsolution to prepare a 100 μM solution, and a 2 μM solution was preparedfrom the 100 μM solution. The 2 μM S1C(disialo)-SRIF28 solution obtainedwas serially diluted with the assay buffer for standard solutionpreparation to prepare standard solutions of 20 nM, 10 nM, 2 nM, 0.4 nM,0.08 nM, and 0.04 nM. By multiplying the results obtained and thedilution ratio, and further multiplying the dilution ratio 2 in theaprotinin-containing EDTA-PBS solution used as anticoagulant treatment,the plasma concentration was calculated. As a control, a similaroperation was carried out employing non-glycosylated SRIF28 instead ofthe glycosylated form. The transition of plasma S1C(disialo)-SRIF28concentration obtained is shown in FIG. 2.

68-4 Estimation of Pharmacokinetics Parameter

From the transition of S1C(disialo)-SRIF28 concentration obtained, AUCwas calculated by the moment analysis method and the trapezoidal rule.Moreover, the predicted initial concentration (C₀) was determined by theextrapolation method for intravenous administration, t_(1/2) and MRTwere calculated, and the maximum plasma concentration (C_(max)) wasdetermined from the actual value for subcutaneous administration. Thepharmacokinetics parameters obtained are shown in Table 4.

[Table 14]

TABLE 4 Intravenous administration Subcutaneous administration t_(1/2)AUC MRT C₀ t_(1/2) AUC MRT C_(max) S1C(disialo)- 18.1 5086 20.4 780 26.01273 43.8 19.9 SRIF28 SRIF28 0.8 358 2.3 678 1.5 15 3.2 4.4 (t_(1/2):min

 AUC: min

 nM

 MRT: min

 C₀: nM

 C_(max): nM)

indicates data missing or illegible when filed

As is clear from the results shown in FIG. 2 and Table 4,S1C(disialo)-SRIF28 has significantly extended t_(1/2) and MRT comparedto the non-glycosylated SRIF28, and an increase in AUC and C_(max) wasrecognized. These are thought to be due to the increase in resistance todegradation activity in blood by glycosylation. It is clear that theglycosylated form has improved stability in vivo compared to thenon-glycosylated form. Moreover, as factors for the increase in AUC andC_(max), the improvement of bioavailability according to the presentinvention, as well as improvement of stability thereof in vivo arethought to be factors.

Example 69 Pharmacokinetics Test with Rats 2

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, andS1C(disialo).N5C(disialo)-SRIF28 were employed as the glycosylatedforms. As a control, non-glycosylated SRIF28 was employed instead of theglycosylated form. The compound plasma concentration transition obtainedis shown in FIG. 3.

Example 70 Pharmacokinetics Test with Rats 3

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, S1C(disialo).R13C(disialo)-SRIF28, andS1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 were employed as theglycosylated forms. As a control, non-glycosylated SRIF28 was employedinstead of the glycosylated form. The compound plasma concentrationtransition obtained is shown in FIG. 4.

Example 71 Pharmacokinetics Test with Rats 4

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28,S1C(disialo).N5C(disialo)-SRIF28, N5C(disialo).A9C(disialo)-SRIF28, andS1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 were employed as theglycosylated forms. The compound plasma concentration transitionobtained is shown in FIG. 5.

Example 72 Pharmacokinetics Test with Rats 5

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, andS1-3C(disialo)-SRIF28 were employed as the glycosylated forms. Thecompound plasma concentration transition obtained is shown in FIG. 6.

From the compound plasma concentration transition obtained in thepharmacokinetics tests of Examples 69-72, the pharmacokineticsparameters of each compound were calculated similarly to Example 68.Moreover, the parameters obtained were employed to calculatebioavailability from the following mathematical formula. The results areshown in 0 Table 5A.

BA (%)=(AUC _((sc))/Dose_((sc)))/(AUC _((iv))/Dose_((iv)))

AUC_((sc)): AUC for subcutaneous administration (min·nM)

Dose_((sc)): Administration dose for subcutaneous administration(nmol/kg)

AUC_((iv)): AUC for intravenous administration (min·nM)

Dose_((iv)): Administration dose for intravenous administration(nmol/kg)

TABLE 5A The number of Value Intravenous administration Subcutaneousadministration Compound sugar chains of n t_(1/2) AUC MRT C₀ t_(1/2) AUCMRT C_(max) BA — SRIF28 None 6 1.00 ± 472 ± 2.20 ± 554 ± 2.94 ± 25.5 ±4.76 ± 4.47 ± 5 0.20 166 0.11 221 1.59 13.8 1.80 1.21 1S1C(disialo)-SRIF28 1 15 16.9 ± 4855 ± 18.6 ± 686 ± 29.8 ± 1773 ± 47.9 ±28.9 ± 37 1.02 1030 1.72 363 2.68 281 2.68 3.23 2 N5C(disialo)-SRIF28 16 20.1 ± 4421 ± 22.9 ± 496 ± 28.1 ± 2392 ± 51.9 ± 32.4 ± 54 1.49 13662.16 312 3.72 443 5.92 4.87 3 A9C(disialo)-SRIF28 1 3 19.9 ± 4083 ± 25.2± 547 ± 30.3 ± 2462 ± 55.6 ± 30.2 ± 60 0.33 515 0.38 211 2.45 64.0 5.815.05 Average value 19.0 4453 22.2 576.3 29.4 2209 51.8 30.5 50 20S1-2C(disialo)-SRIF28 2 3 28.8 ± 7306 ± 23.3 ± 1382 ± 40.9 ± 4046 ± 67.9± 46.3 ± 55 (dense) 3.11 625 2.63 321 12.2 248 15.0 9.75 21 S1C(disialo)

N5C(disialo)-SRIF28 2 6 25.8 ± 4099 ± 24.6 ± 558 ± 38.4 ± 3344 ± 67.9 ±35.5 ± 82 (with intervals) 3.41 634 5.97 248 4.93 663 8.39 6.38 22S1C(disialo)

R13C(disialo)-SRIF28 2 3 23.4 ± 4029 ± 20.6 ± 822 ± 39.6 ± 3407 ± 67.7 ±38.7 ± 85 (with intervals) 0.66 184 1.32 106 2.50 436 5.39 8.73 23N5C(disialo)

A9C(disialo)-SRIF28 2 3 32.4 ± 4465 ± 26.6 ± 664 ± 42.7 ± 3450 ± 75.0 ±32.5 ± 77 (with intervals) 1.66 833 0.27 104 9.76 784 12.4 7.06 Averagevalue 27.2 4198 23.9 681.3 40.2 3400 70.2 35.6 81 24S1-3C(disialo)-SRIF28 3 6 38.5 ± 5660 ± 24.0 ± 1061 ± 87.7 ± 3884 ± 126± 27.5 ± 69 (dense) 2.83 1354 3.92 209 21.4 543 31.7 10.8 25S1C(disialo)

N5C(disialo)

3 9 39.8 ± 4402 ± 25.8 ± 938 ± 87.4 ± 4015 ± 132 ± 24.8 ± 91A9C(disialo)-SRIF28 (with intervals) 1.87 999 2.87 145 17.1 944 19.89.03 (t_(1/2): min, AUC: min

 nM, MRT: min, C₀: nM, C_(max): nM, BA: %)

indicates data missing or illegible when filed

As is clear from the results shown in Table 5A, the bioavailability ofthe glycosylated form of the present invention increased as the numberof modifying sugar chains increased. In other words, it was found thatwhat was 5% in the non-glycosylated form was 37-60% in the glycosylatedpolypeptide having one sugar chain added, an increase of 50% on average,and this was increased to 77-85%, 81% on average in those having twosugar chains added with intervals, and to 91% in those having threesugar chains added with intervals. Moreover, comparing those with denseglycosylation intervals, it was found that there was a 37% increase inthose having one added, 55% in those having two added, 69% in thosehaving three added (the compounds of Examples 1, 20, and 24). From theseresults, it was proven that bioavailability is improved by glycosylationaccording to the present invention.

As factors for the increase in bioavailability for subcutaneousadministration, various pharmacokinetic factors exist. Among thesefactors, some are thought to be the stability of the compound in blood,or transitivity into the blood (from the administration site). As shownin Table 5A, it is recognized that the AUC for subcutaneousadministration which will be an indicator for speculating the transit toblood for subcutaneous administration will increase with the increase inthe number of modifying sugar chains (1: 2209 min·nM, 2 (withintervals): 3400 min·nM, and 3 (with intervals): 4015 min·nM), and itwas speculated to contribute to improvement bioavailability.

Moreover, it was recognized that as the number of modifying sugar chainsincreased, t_(1/2) and MRT extension effect for intravenous andsubcutaneous administrations (for example, t_(1/2) for intravenousadministration was 1: 19.0 min, 2 (with intervals): 27.2 min, and 3(with intervals): 39.8 min), and it was speculated that stability inblood was improved. On the other hand, AUC for intravenousadministration was not an increase in proportion to the number ofmodifying sugar chains (1: 0.4453 min·nM, 2 (with intervals): 4198min·nM, and 3 (with intervals): 4402 min·nM), and it is thought that theincrease in the stability in blood of the compound by the increase inthe number of modifying sugar chains is not the only factor contributingto the increase in bioavailability.

C_(max) for subcutaneous administration increased compared to thenon-glycosylated form up until the number of modifying sugar chains wastwo (0: 4.47 nM, 1: 30.5 nM, and 2 (with intervals): 35.6 nM), and three(with intervals) (24.8 nM) resulted in an adverse decrease. Whenconsidering blood transit from the administration site (subcutaneousadministration in the present Example), speculated factors for theincrease in AUC are two formats of rapid transit to blood, or mild butcontinuous transit to blood. The former has the advantage of avoidingdegradation at the administration site, but if there is no problem instability, it is speculated that the latter format has a higher transitto blood. The fact that C_(max) had decreased in those having threemodifying sugar chains with high bioavailability is thought to be theresult showing not a sudden but a mild and continuous migration inblood. From this, it is thought that the increase in the number ofmodifying sugar chains related to the present invention shows acontinuous migration in blood without a rapid rise in plasmaconcentration (may generally be referred to as absorption delayingeffect).

As is clear from the results shown in Table 5A, there was no significantdifference in t_(1/2) and MRT when the modifying positions had intervalsand when they were dense (for example, t_(1/2) for intravenousadministration was 2 (dense): 28.8 min, 2 (with intervals): 27.2 min, 3(dense): 38.5 min, and 3 (with intervals): 39.8 min).

Moreover, with respect to AUC, for intravenous administration, AUC wasincreased when glycosylation was dense. In other words, for two sugarchains, this was 4029-4465 min·nM for compounds 21, 22, and 23 withintervals as opposed to 7306 min·nM for a dense compound 20, and forthree sugar chains, compound 25 with intervals was 4402 min·nM whereas adense compound 24 was 5660 min·nM.

On the other hand, bioavailability increased when there were intervalsin the glycosylation positions compared to when they were dense. Inother words, for two sugar chains, a dense compound 20 was 55% whereascompounds 21, 22, and 23 with intervals were 77-85% (81% on average),and for three sugar chains, a dense compound 24 was 69% whereas compound25 with intervals was 91%. From this, it is thought that forglycosylation positions related to the present invention, multiplemodifications with intervals contribute more to the improvement ofbioavailability than dense multiple modifications.

Example 73 Pharmacokinetics Test with Rats 7

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, S1C(disialo(amide))-SRIF28, andS1C(disialo(aminoethylamide))-SRIF28 were employed as the glycosylatedforms. The compound plasma concentration transition obtained is shown inFIG. 7.

Example 74 Pharmacokinetics Test with Rats 8

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, S1C(disialo(Bn))-SRIF28, andS1C(disialo)-D-Trp22-SRIF28 were employed as the glycosylated forms. Thecompound plasma concentration transition obtained is shown in FIG. 8.

Example 75 Pharmacokinetics Test with Rats 9

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, R13C(disialo)-SRIF28, and K14C(disialo)-SRIF28were employed as the glycosylated forms. The compound plasmaconcentration transition obtained is shown in FIG. 9.

Example 76 Pharmacokinetics Test with Rats 10

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, E12C(disialo)-SRIF28, N19C(disialo)-SRIF28,29C(disialo)-SRIF28, S1C(monosialo)-SRIF28, and S1C(asialo)-SRIF28 wereemployed as the glycosylated forms. The compound plasma concentrationtransition obtained is shown in FIG. 10.

Example 77 Pharmacokinetics Test with Rats 11

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, K14C(disialo)-SRIF28, and C(disialo)-SRIF14were employed as the glycosylated forms. The compound plasmaconcentration transition obtained is shown in FIG. 11.

Example 78 Pharmacokinetics Test with Rats 12

A pharmacokinetics test was carried out similarly to Example 68, exceptthat C(disialo)-SRIF14, C(disialo)-C12 linker-SRIF14, and C(disialo)-PEGlinker-SRIF14 were employed as the glycosylated forms. The compoundplasma concentration transition obtained is shown in FIG. 12.

Example 79 Pharmacokinetics Test with Rats 13

A pharmacokinetics test was carried out similarly to Example 68, exceptthat SRIF28, S1C(disialo)-SRIF28, S1C(asialo)-SRIF28,S1C(diGlcNAc)-SRIF28, S1C(diMan)-SRIF28, and S1C(GlcNAc)-SRIF28 wereemployed as the glycosylated forms. The compound plasma concentrationtransition obtained is shown in FIG. 13.

Example 80 Pharmacokinetics Test with Rats 14

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, S1C(tetrasialo)-SRIF28, S1C(trisialo)-SRIF28,S1C(Asn(disialo))-SRIF28, and S1-2C(disialo)-SRIF28 were employed as theglycosylated forms. The compound plasma concentration transitionobtained is shown in FIG. 14.

Example 81 Pharmacokinetics Test with Rats 15

A pharmacokinetics test was carried out similarly to Example 68, exceptthat SRIF28, S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28,S1-3C(disialo)-SRIF28, and S1-4C(disialo)-SRIF28 were employed as theglycosylated forms. The compound plasma concentration transitionobtained is shown in FIG. 15.

Example 82 Pharmacokinetics Test with Rats 16

A pharmacokinetics test was carried out similarly to Example 68, exceptthat SRIF14, C(disialo)-SRIF14, C(disialo)-K-SRIF14,C(disialo)-R-K-SRIF14, 2C(disialo)-R-K-SRIF14, and3C(disialo)-R-K-SRIF14 were employed as the glycosylated forms. Thecompound plasma concentration transition obtained is shown in FIG. 16.

Example 83 Pharmacokinetics Test with Rats 17

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28, and S1-3C(asialo)-SRIF28were employed as the glycosylated forms. The compound plasmaconcentration transition obtained is shown in FIG. 17.

Example 84 Pharmacokinetics Test with Rats 18

A pharmacokinetics test was carried out similarly to Example 68, exceptthat S1C(disialo)-SRIF28, S1-2C(disialo)-SRIF28, S1-2C(asialo)-SRIF28,C(disialo(aminoethylamide)/S1C(disialo)-SRIF28,S1-2C(disialo(Bn))-SRIF28, and S1-2C(disialo(amide))-SRIF28 wereemployed as the glycosylated forms. The compound plasma concentrationtransition obtained is shown in FIG. 18.

From the compound plasma concentration transition obtained inpharmacokinetics tests 7-18 of Examples 73-84, the pharmacokineticsparameters of each compound were calculated similarly to Example 68. Thepharmacokinetics parameters obtained are shown in Tables 5B-5F.

TABLE 5B Intravenous administration Subcutaneous administration ExampleT_(1/2) AUC MRT C₀ T_(1/2) AUC MRT Cmax BA — SRIF14 0.500 119 2.00 8192.30 8 4.30 1.80 7 — SRIF28 1.32 467 2.46 518 2.38 22 4.18 4.65 5 1S1C(disialo)-SRIF28 17.7 5190 19.1 633 31.2 1946 49.2 30.1 38 4E12C(disialo)-SRIF28 19.5 10521 27.5 1086 38.2 2891 57.5 34.7 27 5R13C(disialo)-SRIF28 20.3 4926 22.8 481 35.4 5528 55.9 65.9 78 6K14C(disialo)-SRIF28 20.4 6278 20.0 884 35.1 3945 55.1 57.5 62 10N19C(disialo)-SRIF28 18.8 3854 21.5 407 37.9 2624 55.3 41.7 56 1329C(disialo)-SRIF28 23.7 3736 25.3 359 39.3 2176 57.6 29.4 51 15S1C(disialo)-D-Trp-SRIF28 18.3 4924 20.6 588 31.0 2220 51.9 34.0 45 18C(disialo)-R-K-SRIF14 18.9 6571 22.5 429 28.4 1996 45.2 32.5 30 40C(disialo)-K-SRIF14 18.8 5277 19.4 646 26.4 2446 43.7 40.5 46 17C(disialo)-SRIF14 16.5 5614 18.3 725 27.2 3641 40.0 73.6 66 19C(disialo)-C12linker-SRIF14 11.0 1686 7.60 500 14.4 345 22.0 13.4 21 43C(disialo)-PEGlinker-SRIF14 16.4 3538 18.8 412 20.7 2725 39.2 47.8 77

As is clear from the results shown in Table 5B, S1C(disialo)-SRIF28,E12C(disialo)-SRIF28, R13C(disialo)-SRIF28, K14C(disialo)-SRIF28,N19C(disialo)-SRIF28, and 29C(disialo)-SRIF28,S1C(disialo)-D-Trp22-SRIF28 extended t_(1/2) by 13-18-folds andincreased AUC by 8-23-folds compared to the non-glycosylated form SRIF28when administered intravenously. Moreover, C(disialo)-SRIF14,C(disialo)-K-SRIF14, and C(disialo)-R-K-SRIF14 extended t_(1/2) by33-38-folds and increased AUC by 44-55-folds compared to thenon-glycosylated form SRIF14 when administered intravenously. Moreover,C(disialo)-C12 linker-SRIF14 and C(disialo)-PEG linker-SRIF14 extendedt_(1/2) by 22-33-folds and increased AUC by 14-30-folds compared to thenon-glycosylated form SRIF14. In other words, similarly to Example 68,this was thought to be the result of improved stability in blood byglycosylation.

TABLE 5C Intravenous administration Subcutaneous administration ExampleT_(1/2) AUC MRT C₀ T_(1/2) AUC MRT Cmax BA — SRIF28 1.32 467 2.46 5182.38 22 4.18 4.65 5 53 S1C(GlcNAc)-SRIF28 1.80 690 3.00 666 5.90 63 7.907.10 9 51 S1C(diMan)-SRIF28 5.50 1445 5.20 423 9.10 124 13.9 7.30 9 50S1C(diGlcNAc)-SRIF28 10.5 3588 12.3 495 16.5 445 25.7 13.2 12 27S1C(asialo)-SRIF28 13.4 4802 12.5 749 19.7 710 29.0 17.2 16 26S1C(monosialo)-SRIF28 15.9 6618 15.3 1061 24.5 995 37.8 19.4 17 1S1C(disialo)-SRIF28 17.7 5190 19.1 633 31.2 1946 49.2 30.1 38 55S1C(trisialo)-SRIF28 21.1 4428 22.0 423 36.6 1979 56.9 24.9 45 56S1C(tetrasialo)-SRIF28 20.9 4851 23.0 471 40.5 2624 59.4 32.2 54 63S1C(Asn(disialo))-SRIF28 27.9 6488 35.9 365 34.0 2935 55.3 33.9 45 57S1C(disialo(aminoethylamide))-SRIF28 18.6 3473 12.1 746 27.1 284 33.29.60 8 58 S1C(disialo(amide)-SRIF28 18.0 5814 12.6 1048 30.0 961 42.519.1 17 59 S1C(disialo(Bn))-SRIF28 14.4 3708 14.5 526 45.5 1047 52.618.7 28

As is clear from the results shown in Table 5C, when the size of themodifying sugar chain is altered to GlcNAc (monosaccharide), diMan (5sugars), diGlcNAc (7 sugars), asialo (9 sugars), monosialo (10 sugars),disialo (11 sugars), trisialo (14 sugars), and tetrasialo (17 sugars),the t_(1/2), AUC, and bioavailability when administered subcutaneouslyincreased to 2-17-folds, 3-120-folds, and 2-11-folds, respectively,according to the size of the modifying sugar chain. From this, it becameclear that altering the size of the modifying sugar chain not onlyimproved the stability in blood, but allowed change in its increaserate. Moreover, since it is known that dimannose or asialo sugar chainsetc. among these sugar chains interact with particular proteins, theyare thought to be utilizable for targeting a particular protein ororgans and cells having the particular protein. Moreover, asmodification of the sialic acid at the non-reducing terminal,S1C(disialo(amide))-SRIF28, S1C(disialo(aminoethylamide))-SRIF28, andS1C(disialo(Bn))-SRIF28 having added a sugar chain with altered chargeby e.g. the introduction of an aminoethylamide, amide, and Bn to thecarboxy group extended t_(1/2) by 11-14-folds and increased AUC by7-12-folds, and improved stability in blood compared to thenon-glycosylated form when administered intravenously. This shows thatthe carboxy group of the sialic acid has a large influence onretentivity in blood, and shows the possibility for (usage in) bloodclearance or body distribution control by abstraction of the negativecharge of the carboxy group (disialo(Bn) and disialo(amide)) orconversion into a positive charge (disialo(aminoethylamide)).

[Table 18]

TABLE 5D Intravenous administration Subcutaneous administration ExampleT_(1/2) AUC MRT C₀ T_(1/2) AUC MRT Cmax BA — SRIF14 0.500 119 2.00 8192.30 8 4.30 1.80 7 — SRIF28 1.32 467 2.46 518 2.38 22 4.18 4.65 5 18C(disialo)-R-K-SRIF14 18.9 6571 22.5 429 28.4 1996 45.2 32.5 30 482C(disialo)R-K-SRIF14 31.7 5041 24.1 704 47.7 3310 73.4 36.7 66 493C(disialo)R-K-SRIF14 35.7 4288 22.2 1013 70.6 3515 112 23.5 82 1S1C(disialo)-SRIF28 17.7 5190 19.1 633 31.2 1946 49.2 30.1 38 20S1-2C(disialo)-SRIF28 28.2 5804 23.0 950 40.8 3091 65.3 34.6 53 24S1-3C(disialo)-SRIF28 39.5 5834 23.8 1083 79.3 4174 115 30.7 72 66S1-4C(disialo)-SRIF28 63.5 7070 25.2 1007 119 4554 172 22.0 64

As is clear from the results shown in Table 5D, C(disialo)-R-K-SRIF14modified with one disialosugar chain, 2C(disialo)-R-K-SRIF14 modifiedwith two disialosugar chains, and 3C(disialo)-R-K-SRIF14 modified withthree disialosugar chains on SRIF14 each extended t_(1/2) by 38, 63, and71-folds and increased AUC by 55, 42, and 36-folds compared to thenon-glycosylated form when administered intravenously. Similarly,S1C(disialo)-SRIF28 modified with one disialosugar chain,S1-2C(disialo)-SRIF28 modified with two disialosugar chains,S1-3C(disialo)-SRIF28 modified with three disialosugar chains, andS1-4C(disialo)-SRIF28 modified with four disialosugar chains on SRIF28each extended t_(1/2) by 13, 21, 30, and 48-folds and increased AUC by11, 12, 12, and 15-folds compared to the non-glycosylated form whenadministered intravenously. In either of SRIF14 and SRIF28, it becameclear that stability in blood is improved according to the number ofmodifying disialosugar chain.

[Table 19]

TABLE 5E Intravenous administration Subcutaneous administration ExampleT_(1/2) AUC MRT C₀ T_(1/2) AUC MRT Cmax BA — SRIF28 1.32 467 2.46 5182.38 22 4.18 4.65 5 27 S1C(asialo)-SRIF28 13.4 4602 12.5 749 19.7 71029.0 17.2 16 28 S1-2C(asialo)-SRIF28 8.30 2664 9.60 741 19.2 1049 36.419.8 39 29 S1-3C(asialo)-SRIF28 6.60 2733 6.50 1147 26.4 885 42.1 15.032

As is clear from the results shown in Table 5E, S1C(asialo)-SRIF28modified with one asialosugar chain, S1-2C(asialo)-SRIF28 modified withtwo asialosugar chains, and S1-3C(asialo)-SRIF28 modified with threeasialosugar chains on SRIF28 each extended t_(1/2) by 10, 6, and 5-foldsand increased AUC by 10, 6, and 6-folds compared to the non-glycosylatedform when administered intravenously. It became clear that stability inblood is also improved when the modifying sugar chain is an asialosugarchain.

TABLE 5F Intravenous administration Subcutaneous administration ExampleT_(1/2) AUC MRT C₀ T_(1/2) AUC MRT Cmax BA — SRIF28 1.32 467 2.46 5182.38 22 4.18 4.65 5 1 S1C(disialo)-SRIF28 17.7 5190 19.1 633 31.2 194649.2 30.1 38 20 S1-2C(disialo)-SRIF28 28.2 5804 23.0 950 40.8 3091 65.334.6 53 28 S1-2C(asialo)-SRIF28 8.30 2664 9.60 741 19.2 1049 36.4 19.839 61 S1-2C(disialo(amide))-SRIF28 23.8 5268 20.5 737 33.1 1297 55.816.3 25 62 S1-2C(disialo(Bn))-SRIF28 25.6 5525 20.7 807 33.9 1843 59.421.5 33 65 C(disialo(aminoethylamide))

S1C(disialo)-SRIF28 22.5 3742 20.5 518 42.3 1289 64.7 16.2 34

indicates data missing or illegible when filed

As is clear from the results shown in Table 5F, S1-2C(disialo)-SRIF28,S1-2C(asialo)-SRIF28, C(disialo(aminoethylamide))/S1C(disialo)-SRIF28,S1-2C(disialo(Bn))-SRIF28, and S1-2C(disialo(amide))-SRIF28 eachextended t_(1/2) by 6-21-folds and increased AUC by 6-12-folds comparedto the non-glycosylated form when administered intravenously. In otherwords, stability in blood improved by modifying a sugar chain ontoSRIF28. Moreover, t_(1/2), AUC, Cmax, and BA increased in bothintravenous and subcutaneous administrations according to the increasein the number of sialic acid sugar chains. On the other hand, when thenegative charge of the carboxy group of the sialic acid was removed(2C(disialo(Bn))/2C(disialo(amide))), or when a positive charge wasadjacently added (C(disialo(aminoethylamide))/S1C(disialo)), althought_(1/2) was extended when administered subcutaneously, AUC or Cmax didnot increase. Since the carboxy group of the sialic acid has a largeinfluence on retentivity in blood or blood migration, this shows thepossibility for (usage in) blood clearance or body distribution controlby conversion of charge at the sugar chain terminal.

Example 85 Plasma Stability Test with Rat Plasma 85-1 Preparation ofCompound Solution, Reagent, and Rat Plasma

The glycosylated form and the non-glycosylated SRIF28 were dissolved inPBS solution to prepare 2 μM solutions as treatment solutions. 10% TFAwas prepared by dissolving trifluoroacetic acid (208-02746 from WakoPure Chemical Industries, Ltd.) in water to 10 v/v %. Rat plasma wasprepared from Wistar rats (Crlj: Wistar, male, Charles River Japan, 7weeks-old) as heparin-added plasma (heparin: Japanese Pharmacopeiaheparin sodium injectable solution (Mochida Pharmaceutical Co., Ltd.)).

85-2 Preparation of Plasma-Added Sample

To 0.27 mL of rat plasma (n=3), 0.03 mL of the glycosylated compoundsolution prepared in the above 85-1 was promptly mixed as plasma-addedsample, and kept warm in a 37° C. thermostat bath. After mixing, 0.04 mLof plasma-added sample was taken at 0 minute and over time at 1-24hours, and then promptly mixed with 0.01 mL of 10% TFA. Aftercentrifugal separation (20,000×g, 4° C., 10 minutes), 0.04 mL of thesupernatant was taken as plasma stability measurement samples. Samplingtime was 0, 1, 2, and 4 hours, or 0, 4, 8, and 24 hours. As blankplasma, plasma obtained from a similar treatment except that PBSsolution was employed as the treatment solution was employed. Plasmasamples were frozen in storage until employed for measurement. For eachexperiment run, non-glycosylated SRIF28 was employed as the positivecontrol.

85-3 Measurement of Concentration in Sample and Calculation of PlasmaStability Index

In a method similar to plasma concentration measurement of Example 68-3,the concentration of the glycosylated form remaining in the plasmastability measurement samples obtained in the above 85-2 was measured.The concentration of the glycosylated form at 0 minute after mixing wasset as 100%, the residual rate over time was represented in percentage(%), and the half-life was calculated from the elimination rate constantof the following exponential formula (1) employing the calculationformula (2). Then, the half-life of the non-glycosylated SRIF28 for eachexperiment run was set as 1, and the plasma stability index (PS index)of the glycosylated form was calculated. The results are shown in Table6 and FIG. 19. Moreover, those having a plasma stability index of 20 orhigher are shown as >20. In FIG. 19, those having a plasma stabilityindex of higher than 20 are also shown with 20 as the upper limit.

Residual Rate (%)=100·e ^((k·t))  (1)

e: Base of natural logarithm

k: elimination rate constant

t: Time (hours)

Half-life (hours)=0.693/k  (2)

[Table 21]

TABLE 6 Example PS index 1 S1C(disialo)-SRIF28 4.5 2 N5C(disialo)-SRIF284.7 3 A9C(disialo)-SRIF28 6.7 4 E12C(disialo)-SRIF28 6.2 5R13C(disialo)-SRIF28 15.9 6 K14C(disialo)-SRIF28 19.1 10N19C(disialo)-SRIF28 >20 13 29C(disialo)-SRIF28 >20 1430C(disialo)-SRIF28 16.8 15 S1C(disialo)-D-Trp-SRIF28 12.3 16A9C(disialo)-D-Trp-SRIF28 17.2 21 S1C(disialo)•N5C(disialo)-SRIF28 17.822 S1C(disialo)•R13C(disialo)-SRIF28 >20 24 S1-3C(disialo)-SRIF28 >20 25S1C(disialo)•N5C(disialo)•A9C(disialo)-SRIF28 >20 26S1C(monosialo)-SRIF28 3.5 27 S1C(asialo)-SRIF28 2.6 — Octreotide >20 —SRIF28 1

As is clear from the results shown in FIG. 19, the glycosylatedpolypeptide of the present invention was increased in plasma stabilitycompared to SRIF28. In other words, this was 4.5-6.7-folds in thosehaving one sugar chain added at positions 1, 5, 9, and 12 (the compoundsof Examples 1, 2, 3, and 4), 15.9-folds for those having a sugar chainadded at position 13 (the compound of Example 5), 19.1-folds for thosehaving a sugar chain added at position 14 (the compound of Example 6),and 16.8-folds for those having a sugar chain added at position 30 (thecompound of Example 14), compared to that of SRIF28. Moreover, this was20-folds or more for those having one glycosylated amino acid furtheradded at position 19 which is the C-terminal side (the compounds ofExamples 10 and 13). It was shown that in regards to the position forglycosylation, plasma stability increases from position 1 towards theC-terminal.

Example 86 GH Production Suppression Test with Rats

As shown in Example 67, the glycosylated polypeptide of the presentinvention had affinity towards each receptor of SSTR. On the other hand,some were seen to have attenuated affinity towards each SSTR, but inorder to prove that even in such cases pharmacologically effectiveaction is shown towards receptors in vivo by e.g. extension of half-lifein blood or increase in bioavailability as shown in Examples 68-85, atest for evaluating the administration effect of the glycosylatedpolypeptide of the present invention on growth hormone (GH) productionwas carried out as an in vivo test employing rats. Increase of GHproduction amount into the blood is thought to cause proliferation ordifferentiation of cells and activation or suppression of thebiosynthetic system in various organs via the paracrine effect of GH,and thereby influence biological reactions. Somatostatin released fromthe hypothalamus suppresses GH secretion from the anterior pituitarygland into the blood. This experimental line was carried out as a testline that can evaluate the pharmacological action of the glycosylatedform on SSTR and its residence in blood after administration, with bloodGH production amount as an indicator.

86-1 Preparation of Administration Solution and Reagent

Employing S1C(disialo)-SRIF28, N19C(disialo)-SRIF28, and29C(disialo)-SRIF28 as the glycosylated forms, these were dissolved inJapanese Pharmacopeia saline (from Otsuka Pharmaceutical factory, Inc.)to prepare 1-100 μM solutions as the administration solution. Moreover,GRF (GH releasing hormone, Growth hormone releasing factor) was used asthe GH release enhancer. GRF was prepared by dissolving GRF injectablesolution (GRF Sumitomo 50 for injection, Lot. 2006C, from DainipponSumitomo Pharma Co., Ltd.) in 1 mL of water for injection (Lot. 09H18C,from Fuso Pharmaceutical Industries, Ltd.), and then diluting to25-folds with saline to obtain 2 μg/mL. As heparin employed as theanticoagulant when collecting blood, Japanese Pharmacopeia heparinsodium injectable solution (Lot. B084, from Mochida Pharmaceutical Co.,Ltd.) was used directly as stock solution.

86-2 Preparation of Plasma Sample

To the dorsal subcutaneous of male SD rats (Crl: CD (SD), Charles RiverJapan, 6 weeks-old, n=3, body weight 145.2-163.9 g), the administrationsolution prepared in the above 86-1 was administered under nonfastingcondition at a dosage of 1 mL/kg with a glass syringe and a 26 Ginjection needle (all from Terumo Corporation). As the control group,saline without glycosylated polypeptide was similarly administered(vehicle group). Then, i.e. between 5-6 minutes after the administrationof the glycosylated form, pentobarbital sodium (somnopentyl, Lot.0608101, from Kyoritsuseiyaku Corporation) was intraperitoneallyadministered as a general anesthetic with a glass syringe and aninjection needle to give 50 mg/kg. One hour after administration of theglycosylated form, i.e. after 50 minutes or more had passed underanesthesia, GRF was administered as the GH release enhancer to the tailvain at a dosage of 1 mL/kg with a glass syringe and an injectionneedle. Five minutes after administration of GRF, blood was collectedfrom the rat cervical vein with a glass syringe containing heparin andan injection needle. 0.4 mL of the collected blood was left on ice for20 minutes or more, then centrifuged (1,870×g, 4° C., 10 minutes), and100 μL of the supernatant was taken as the plasma sample. As blankplasma, plasma obtained by similarly treating the blood collected fromuntreated rat cervical vein was employed. Plasma samples were frozen instorage until employed for measurement.

86-3 Measurement of GH Concentration in Plasma

Measurement of GH concentration in the plasma sample obtained in theabove 86-2 was performed with rat Growth Hormone EIA kit from SPI-Bio(SPI-Bio, A05104). The plasma sample was diluted with the assay buffersupplied in the EIA kit to 20, 100, and 500-folds as measurementsamples. The standard solution for creating a standard curve followedthe attached instructions by preparing a 40 ng/mL solution withdistilled water, and then subjecting to serial dilution with the assaybuffer to prepare 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL,0.63 ng/mL, and 0.31 ng/mL. By multiplying the results obtained and thedilution ratio, the GH concentration was calculated. The GHconcentration in the plasma sample obtained is shown in Table 7.

[Table 22]

TABLE 7 Ex- GH am- (ng/ ple Dose mL) 1 S1C(disialo)-SRIF28 1 nmol/kg 4901 S1C(disialo)-SRIF28 10 nmol/kg 17.1 10 N19C(disialo)-SRIF28 10 nmol/kg563 10 N19C(disialo)-SRIF28 100 nmol/kg 1.5 13 29C(disialo)-SRIF28 10nmol/kg 497 13 29C(disialo)-SRIF28 100 nmol/kg 88.5 21S1C(disialo)•N5C(disialo)-SRIF28 1 nmol/kg 776 21S1C(disialo)•N5C(disialo)-SRIF28 10 nmol/kg 181 25S1C(disialo)•N5C(disialo)•A9C(disialo)- 10 nmol/kg 833 SRIF28 25S1C(disialo)•N5C(disialo)•A9C(disialo)- 100 nmol/kg 151 SRIF28 — vehicle— 861

As is clear from the results shown in Table 7, the glycosylated form ofthe present invention suppressed GH production in rats. The ratiobetween the Ki value of glycosylated forms of Example 1, Example 10, andExample 13 against each receptor and the Ki value of unglycosylatedSRIF14 is shown to be in the range of 100:1-1.3:1, as measured in themethod of Example 67-1. Moreover, the glycosylated form of Example 1 isshown to have 10-folds or more increase in half-life in blood, asmeasured in the method of Examples 68-72. From the present Example, itwas shown that the glycosylated form effectively exerts pharmacologicalaction even in vivo.

Moreover, the glycosylated form of the present invention had GHproduction suppressing action even when administered 1 hour before GRFadministration.

Moreover, it was shown that the effective administration dose for rat GHproduction ability between the glycosylated forms of Example 1, Example10, and Example 13 was different by approximately 10-folds. This issimilar to the receptor affinities thereof having a difference ofapproximately 10-folds in terms of Ki value, as measured in the methodof Example 67-1. It was shown that they have pharmacological activityeven when the affinity of the glycosylated form was somewhat attenuated.

Example 87 GH Production Suppression Test with Rats 2

Similarly to Examples 86-1-86-3, rat GH production suppression tests ofthe glycosylated compounds shown below were carried out.S1C(disialo)-SRIF28, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28,E12C(disialo)-SRIF28, R13C(disialo)-SRIF28, K14C(disialo)-SRIF28,S1C(disialo)-D-Trp22-SRIF28, S1C(disialo(Bn))-SRIF28,S1C(disialo(amide))-SRIF28, S1C(disialo(aminoethylamide))-SRIF28,S1C(monosialo)-SRIF28, S1C(asialo)-SRIF28, C(disialo)-R-K-SRIF14,S1-2C(asialo)-SRIF28, SiC (disialo).N5C(disialo)-SRIF28, andS1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 were employed as theglycosylated forms. The GH concentration in the plasma sample obtainedis shown in Table 8.

[Table 23]

TABLE 8 The number Example Dose GH (ng/mL) of runs 1 S1C(disialo)-SRIF281 nmol/kg 787 4 3 nmol/kg 182 3 10 nmol/kg 15 5 30 nmol/kg 8 1 100nmol/kg 7 2 2 N5C(disialo)-SRIF28 1 nmol/kg 969 1 10 nmol/kg 12 1 3A9C(disialo)-SRIF28 1 nmol/kg 577 1 10 nmol/kg 20 1 4E12C(disialo)-SRIF28 10 nmol/kg 10 1 5 R13C(disialo)-SRIF28 10 nmol/kg136 1 6 K14C(disialo)-SRIF28 10 nmol/kg 55 1 15S1C(disialo)-D-Trp22-SRIF28 0.3 nmol/kg 986 3 1 nmol/kg 335 3 3 nmol/kg50 1 10 nmol/kg 6 2 30 nmol/kg 2 1 18 C(disialo)-R-K-SRIF14 1 nmol/kg912 4 3 nmol/kg 291 4 10 nmol/kg 30 4 30 nmol/kg 12 1 21S1C(disialo)•N5C(disialo)-SRIF28 1 nmol/kg 776 1 10 nmol/kg 181 1 25S1C(disialo)•N5C(disialo)•A9C(disialo)-SRIF28 100 nmol/kg 151 1 26S1C(monosialo)-SRIF28 10 nmol/kg 47 1 27 S1C(asialo)-SRIF28 1 nmol/kg879 4 3 nmol/kg 515 4 10 nmol/kg 146 4 28 S1-2C(asialo)-SRIF28 3 nmol/kg997 3 10 nmol/kg 175 3 57 S1C(disialo(aminoethylamide))-SRIF28 10nmol/kg 314 1 58 S1C(disialo(amide))-SRIF28 10 nmol/kg 14 1 59S1C(disialo(Bn))-SRIF28 10 nmol/kg 49 1 — vehicle — 1012 19

As is clear from the results shown in Table 8, the glycosylatedpolypeptide of the present invention suppressed GH production in rats.In the present test line, S1C(disialo)-SRIF28 suppressed GH productionfrom 1 nmol/kg, and showed 82-99% of the GH production suppressioneffect at 3-10 nmol/kg. This is thought to be due to the gain inaffinity towards SSTR1-SSTR5 and improvement in the retentivity in bloodas shown in Examples 67-1, 67-2, and 68-85.

In Examples 67-1 and 67-2, N5C(disialo)-SRIF28, A9C(disialo)-SRIF28,E12C(disialo)-SRIF28, S1C(disialo)-D-Trp-SRIF28,S1C(disialo(Bn))-SRIF28, and C(disialo)-R-K-SRIF14 showing affinityequivalent to S1C(disialo)-SRIF28 showed 95-99% of the GH productionsuppression effect at 10 nmol/kg, showing an effect equivalent toS1C(disialo)-SRIF28.

From the results shown in the methods of Examples 68-85,S1C(monosialo)-SRIF28, S1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28, andS1C(disialo(amide))-SRIF28 had an AUC for subcutaneous administrationwhich was ⅓-½ of S1C(disialo)-SRIF28, andS1C(disialo(aminoethylamide))-SRIF28 was 1/7. Meanwhile, all of theseshowed higher affinity than S1C(disialo)-SRIF28 by the methods shown inExamples 67-1 and 67-2. Accordingly, in this experimental line,S1C(monosialo)-SRIF28, S1C(asialo)-SRIF28, S1-2C(asialo)-SRIF28,S1C(disialo(aminoethylamide))-SRIF28, and S1C(disialo(amide))-SRIF28suppressed 70-99% of the GH production at 10 nmol/kg, and is thought tohave shown an effect equivalent to S1C(disialo)-SRIF28.

R13C(disialo)-SRIF28, K14C(disialo)-SRIF28,S1C(disialo).N5C(disialo)-SRIF28, andS1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 showed a lower affinitythan S1C(disialo)-SRIF28 by the methods shown in Examples 67-1 and 67-2.Meanwhile, from the results shown in the methods of Examples 68-85,these compounds have AUC for subcutaneous administration that wasimproved to 1.8-2.8-folds of S1C(disialo)-SRIF28. In this experimentalline, R13C(disialo)-SRIF28, K14C(disialo)-SRIF28,S1C(disialo).N5C(disialo)-SRIF28, andS1C(disialo).N5C(disialo).A9C(disialo)-SRIF28 showed GH productionsuppression activity by administration of 10 or 100 nmol/kg. Theseresults show that somatostatin activity can be compensated or increasedby the increase in stability in blood even when receptor affinity isreduced.

Example 88 Drug Effect Test in Gastrointestinal Obstruction Model

As shown in Examples 86 and 87, the glycosylated polypeptides of thepresent invention proved to have effective action as agonists even inrats in vivo. Next, in order to prove that they also show efficacy indisease models, an evaluation in rat gastrointestinal obstruction modelwas carried out. In gastrointestinal obstructions such as ileus,gastrointestinal symptoms such as sense of abdominal fullness, vomiting,and abdominal pain are shown by gastrointestinal tract tissue disorderand reduction in absorption ability of e.g. water or electrolyte. Theirpathologies are known to be caused by obstruction of gastrointestinalcontent or release of digestive juice or biologically active materialinto the gastrointestinal tract. Somatostatin show the effect ofdecreasing the gastrointestinal content by secretory suppression ofvarious digestive juices or promotion of water and electrolytesabsorption via SSTR expressed in the gastrointestinal system, and isthought to be effective for improvement of the symptoms. Thisexperimental line was carried out as a test line to evaluate thepromotion of intestinal fluid absorption or the secretory suppressingaction, using the change in the intestinal fluid weight in the jejunumafter bowel obstruction as an indicator. Moreover, blood parameters ofdeviation enzymes amylase (pancreas), lactate dehydrogenase (LDH,liver), and creatine phosphokinase (CPK, skeletal muscle, cardiac muscleetc.) were measured as indicators of tissue disorders.

Example 88-1 Production of Gastrointestinal Obstruction Model

The present test was consigned to Mitsubishi Chemical MedienceCorporation and carried out. Male SD rats (Crl: CD (SD), Charles RiverJapan, 8 weeks-old, n=5 or more, body weight 251.1-278.1 g) were fastedfor 12 hours or more. Anesthesia was introduced with inhalation of 2%isoflurane and laughing gas:oxygen=7:3, and this was maintainedthroughout the surgery. A median incision was made in the abdomen, andthe jejunum at about 10 cm from the musculus suspensorius duodeni wasligated with a surgical suture. Then, the incision site was promptlysutured, and the rats were fasted until compound administration. In thesham treatment group, ligation of the jejunum was not performed, but thetreatment of suturing the incision site after a median incision was madein the abdomen was performed.

Example 88-2 Preparation of Compound and Administration

Employing S1C(disialo)-SRIF28, C(disialo)-R-K-SRIF14, andS1-2C(asialo)-SRIF28 as the glycosylated forms, these were dissolved inJapanese Pharmacopeia saline (from Otsuka Pharmaceutical factory, Inc.)to prepare a 40 μM solution as the administration solution. Eighteenhours after the gastrointestinal obstruction surgery, this wassubcutaneously administered into the dorsal cervix at a dosage of 1mL/kg with a glass syringe and a 25 G injection needle (all from TerumoCorporation). As the vehicle group, saline without glycosylatedpolypeptide was similarly administered. Moreover, octreotide wasadministered as the control.

Example 88-3 Measurement of Intestinal Fluid Weight

One hour after compound administration, a median incision was made inthe abdomen under inhalation anesthesia, and 1.5 mL of blood wascollected from the abdominal vena cava. Then, the ligated jejunum on themusculus suspensorius duodeni side was resected. The fluid and blood onthe jejunum surface were removed with a paper towel, nerves, bloodvessels, and fat attached to the jejunum was removed, this was cut into6 cm lengths, and wet weight was measured. This was then dried at 36degrees for 24 hours, and dry weight was measured. The intestinal fluidweight (mg) was calculated by wet weight−dry weight. The intestinalfluid weight of the jejunum obtained is shown in Table 9.

[Table 24]

TABLE 9 Intestinal fluid weight (mg) Sham treatment 285 ± 27 vehicle 481± 31 octreotide 693 ± 48 S1C(disialo)-SRIF28 630 ± 83C(disialo)-R-K-SRIF14 584 ± 95 S1-2C(asialo)-SRIF28 566 ± 34

As apparent from Table 9, the vehicle had significantly increasedintestinal fluid weight compared to the sham treatment, and it wasrecognized that enhancement of intestinal fluid secretion accompanyinggastrointestinal obstruction due to ligation was caused. Somatostatin oroctreotide are shown to have the secretory suppression effect of theintestinal fluid into the intestinal tract and the promotional effect ofintestinal fluid absorption into the bowel tissue side in suchgastrointestinal obstruction models (Scand. J. Gastroenterol. 1995 May;30 (5): 464-9), and octreotide was confirmed to have this effect also inthis experimental line. S1C(disialo)-SRIF28, C(disialo)-R-K-SRIF14, andS1-2C(asialo)-SRIF28 were all recognized to have increase in theintestinal fluid weight compared to the vehicle, and it became clearthat the glycosylated forms of the present invention also show efficacysuch as secretory suppression of the intestinal fluid and promotion ofwater and electrolyte absorption. Moreover, it has become clear that inExamples 83 and 84, the AUC for subcutaneous administration ofS1-2C(asialo)-SRIF28 was ½ compared to S1C(disialo)-SRIF28, but inExample 67-1, the affinity towards SSTR1-SSTR5 is approximately1.7-2.9-folds higher than S1C(disialo)-SRIF28. This is thought to be thereason why improvement of receptor affinity causes the drug effects inthe present model to be similar even when plasma concentration is low.Similarly, this is thought to be the reason why drug effects are similarbecause receptor affinity is approximately 0.7-2.4-folds higher eventhough C(disialo)-R-K-SRIF14 has a slightly low AUC for subcutaneousadministration compared to that of S1C(disialo)-SRIF28.

Example 88-4 Measurement of Blood Parameter

Employing the blood collected in Example 88-3, amylase (IU/L), LDH(IU/L) and CPK concentrations (IU/L) were measured with an autoanalyzer7170 (Hitachi, Ltd.). The measuring methods employed were BG5B, UV-rate,and JSCC methods, respectively. The results obtained are shown in Table10.

[Table 25]

TABLE 10 Amylase (IU/L) LDH (IU/L) CPK (IU/L) Sham treatment  669 ± 164168 ± 82 353 ± 68 vehicle 1672 ± 743 341 ± 68 377 ± 57 octreotide 1732 ±774 269 ± 141 472 ± 215 S1C(disialo)-SRIF28 1164 ± 224 338 ± 97 455 ±202 C(disialo)-R-K-SRIF14  985 ± 238 228 ± 155 415 ± 137S1-2C(asialo)-SRIF28 1594 ± 573 318 ± 44 728 ± 665

As apparent from Table 10, the vehicle had increased amylase activityand LDH activity compared to the sham treatment, and it was speculatedthat tissue disorder of the gastrointestinal system had developedaccompanying gastrointestinal obstruction. In S1C(disialo)-SRIF28 andC(disialo)-R-K-SRIF14, the amylase activity had a low value compared tothe vehicle. On the other hand, the amylase activity of octreotide wasequivalent to the vehicle. As apparent from Examples 67-1 and 67-2,S1C(disialo)-SRIF28, C(disialo)-R-K-SRIF14, and S1-2C (sialo)-SRIF28 hadbinding affinity toward all the receptors from SSTR1-SSTR5, whereasoctreotide is a compound having specific affinity towards SSTR2, SSTR3,and SSTR5. In rat pancreas, since there is a report that all thereceptors SSTR1-SSTR5 are expressed (J Histochem Cytochem. 2004 March;52 (3): 391-400), the possibility that the glycosylated form alleviatedthe tissue disorder by acting on a receptor different from that ofoctreotide was conceived. Moreover, in octreotide andC(disialo)-R-K-SRIF14, the LDH activity had a low value compared to thevehicle. There were no others that reduced each parameter byadministration of the glycosylated form.

1-35. (canceled)
 36. A glycosylated polypeptide selected from the groupconsisting of: (A) a SRIF14 consisting of the amino acid sequence of SEQID NO:1; (B) a polypeptide having one or a few amino acids deleted from,substituted with, or added to SRIF14, SRIF14 consisting of the aminoacid sequence of SEQ ID NO:1; (C) a SRIF14 analog; (D) a polypeptidehaving 80% or more homology with SRIF14 consisting of the amino acidsequence of SEQ ID NO: 1; (E) a polypeptide further comprising N aminoacids at the N-terminal side of (A)-(D), wherein N is an integer from 1or more to 20 or less at the N-terminal side of (A)-(D); and (F) apolypeptide further comprising M amino acids at the C-terminal side of(A)-(D), wherein M is an integer from 1 or more to 6 or less; wherein atleast one amino acid is substituted with a glycosylated amino acid, andthe polypeptide has affinity towards somatostatin receptors.
 37. Theglycosylated polypeptide of claim 36, further comprising any one of thefollowing features: the amino acid substituted with said glycosylatedamino acid is the amino acid corresponding to position 19 of SRIF14; atleast one of the amino acids substituted with said glycosylated aminoacid is present in any of said N amino acids at the N-terminal side ofsaid polypeptide (E), wherein at least one of the amino acidssubstituted with said glycosylated amino acid is present in any of saidN amino acids at the N-terminal side of said polypeptide (E); or atleast one amino acid is substituted with glycosylated amino acid in saidpolypeptide (F), wherein at least one of the amino acids substitutedwith said glycosylated amino acid is present in any of said M aminoacids at the C-terminal side of said polypeptide (F).
 38. Theglycosylated polypeptide of claim 36, wherein at least one amino acid issubstituted with glycosylated amino acid in said polypeptide (E), andfurther the sequence of said N amino acids added onto the N-terminalside is represented by X—Y—, wherein X is a sequence of any L aminoacids, and L is an integer from 1 or more to 6 or less; and Y is asequence selected from the group consisting of: (1) Lys, (2) Arg-Lys,(3) Glu-Arg-Lys, (4) Arg-Glu-Arg-Lys, (5) Pro-Arg-Glu-Arg-Lys, (6)Ala-Pro-Arg-Glu-Arg-Lys, (7) Met-Ala-Pro-Arg-Glu-Arg-Lys, (8)Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (9)Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (10)Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys, (11)Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg- Lys, (12)Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu- Arg-Lys, (13)Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg- Glu-Arg-Lys, (14)Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro- Arg-Glu-Arg-Lys, and (15)a sequence having one or a few amino acidsdeleted from, substituted with, or addedto the above sequences (2) - (14).


39. The glycosylated polypeptide according to claim 38, wherein at leastone amino acid substituted with said glycosylated amino acid is presentin any of L amino acids and X in X—Y— represents the sequence of said Namino acids added onto the N-terminal side of said polypeptide (E). 40.The glycosylated polypeptide of claim 36, wherein said glycosylatedpolypeptide has one or more of the following properties: (a) saidaffinity towards somatostatin receptors is an affinity towards at leasttwo or more receptors selected from the group consisting of SSTR1,SSTR2, SSTR3, SSTR4, SSTR5 and/or (b) said glycosylated peptide hasincreased stability in blood compared to SRIF28.
 41. The glycosylatedpolypeptide of claim 36, wherein each of said glycosylated amino acidsis glycosylated Asn or glycosylated Cys.
 42. The glycosylatedpolypeptide of claim 36, wherein each of said glycosylated amino acidscomprises a sugar chain comprising 4 or more sugars.
 43. A method fortreating or preventing a somatostatin-related disease, comprisingadministering to a patient in need thereof an effective amount of aglycosylated polypeptide of claim
 1. 44. The method of claim 43, whereinsaid somatostatin-related disease is at least one disease selected fromthe group consisting of acromegaly, gigantism, Alzheimer's disease andother forms of dementia, cancer, hormone-producing tumor, endocrinetumor, carcinoid, VIPoma, insulinoma, glucagonoma, Cushing's disease,hormone secretion defect, diabetes and complications thereof, pains,arthritis, diarrhea, gastric ulcer, inflammatory bowel disease,irritable bowel syndrome, gastrointestinal obstruction, ileus,postoperative restenosis, eye disease, radiation damage, dry eye,glaucoma, interstitial keratitis, iritis, cataract, and conjunctivitis.